and gamma-butyrolactone (GBL) using NMR a

Our reference: FSI 6488
P-authorquery-v9
AUTHOR QUERY FORM
Journal: FSI
Please e-mail or fax your responses and any corrections to:
E-mail: [email protected]
Article Number: 6488
Fax: +353 6170 9272
Dear Author,
Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen
annotation in the PDF file) or compile them in a separate list. To ensure fast publication of your paper please return your
corrections within 48 hours.
For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions.
Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in
the proof. Click on the ‘Q’ link to go to the location in the proof.
Location in
article
Q1
Q2
Query / Remark: click on the Q link to go
Please insert your reply or correction at the corresponding line in the proof
Please check document heading.
Please provide author details for Ref. [16].
Thank you for your assistance.
G Model
FSI 6488 1–6
Forensic Science International xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International
journal homepage: www.elsevier.com/locate/forsciint
1
2
Q1 Short
communication
5
Report on the analysis of common beverages spiked with gamma-hydroxybutyric
acid (GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE
solvent-suppression technique
6
Casey T. Lesar a, John Decatur b, Elaan Lukasiewicz a, Elise Champeil a,*
3
4
7
8
a
b
Department of Science, John Jay College of Criminal Justice, City University of New York, 445 west 59th Street, New York, NY 10019, USA
Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 21 February 2011
Received in revised form 16 June 2011
Accepted 19 June 2011
Available online xxx
In forensic evidence, the identiﬁcation and quantitation of gamma-hydroxybutyric acid (GHB) in
‘‘spiked’’ beverages is challenging. In this report, we present the analysis of common alcoholic beverages
found in clubs and bars spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL).
Our analysis of the spiked beverages consisted of using 1H NMR with a water suppression method called
Presaturation Utilizing Relaxation Gradients and Echoes (PURGE). The following beverages were
analyzed: water, 10% ethanol in water, vodka–cranberry juice, rum and coke, gin and tonic, whisky and
diet coke, white wine, red wine, and beer. The PURGE method allowed for the direct identiﬁcation and
quantitation of both compounds in all beverages except red and white wine where small interferences
prevented accurate quantitation. The NMR method presented in this paper utilizes PURGE water
suppression. Thanks to the use of a capillary internal standard, the method is fast, non-destructive,
sensitive and requires no sample preparation which could disrupt the equilibrium between GHB and
GBL.
ß 2011 Published by Elsevier Ireland Ltd.
Keywords:
PURGE
gamma-Hydroxybutyric acid
gamma-Butyrolactone
Alcoholic beverages
Nuclear Magnetic Resonance
Water suppression technique
9
10
1. Introduction
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
gamma-Hydroxybutyric acid (GHB), a naturally occurring short
chain carboxylic acid, is a central nervous system depressant that
has euphoric and sedative effects [1–3]. GHB was initially used in
anesthesia. Shortly after it appeared on the market, cases of abuse
began to emerge and the drug became especially popular at ‘‘rave’’
parties [4–6]. GHB powder, usually found as the sodium salt
NaGHB, dissolves rapidly in water giving a colorless solution. GHB
abuse exploded in the late 1990s with GHB related emergency
room visits going from 56 recorded visits in 1994, to 4969 visits in
2000 [7]. GHB appears to affect its users differently according to
many factors (weight, metabolism, etc.) [8]. It has been suggested
that a 0.5 g dose be taken for relaxation and disinhibition, a 1 g
dose for euphoric effect and a 2–3 g dose for deep sleep [9–11]. At
higher doses, GHB may induce depressed breathing and even death
[5]. The effects of GHB can last from 3 to 6 h, or longer if a large dose
has been consumed or if mixed with alcohol [6]. Such is the
scenario when GHB is used as a ‘‘sexual assault’’ or ‘‘date rape’’ drug
through combination with alcoholic beverages. In humans, GHB
* Corresponding author. Tel.: +1 6465574502; fax: +1 2122378318.
E-mail address: [email protected] (E. Champeil).
has been shown to inhibit the elimination rate of alcohol. This may
explain the respiratory arrest that has been reported after its
ingestion [11,12].
GHB undergoes intramolecular esteriﬁcation to form the
corresponding lactone known as gamma-butyrolactone (GBL).
GBL is a solvent used in applications such as paint removal and
engine cleaning. Conversely, GBL can easily be hydrolyzed to GHB
in aqueous solutions as well as in the body. This interconversion
creates both forensic and legal problems [13,14]. It was also found
that under acidic conditions GBL will react with ethanol or
methanol to give the corresponding ethyl and methyl esters of GHB
[15]. The ofﬁcial status of both GBL and GHB vary across European
countries and in the United States [16–18]. Legal distinctions
between GHB and GBL exist at the international level [18,19].
Forensic analysis and identiﬁcation of GHB is performed using a
number of different techniques: Raman spectroscopy [20], FT-IR
[21,22], UV–Vis and mass spectrometry [23] and microchemical
analysis [24]. The most common means of identiﬁcation is gas
chromatography coupled with mass spectrometry (GC/MS). GHB
and GBL can also be identiﬁed and quantiﬁed using liquid
chromatography coupled with electrospray ionization mass
spectrometry (LC/MS) [25]. All these techniques have drawbacks.
For instance, GHB detection by GC requires derivatization, such as
conversion to the di-trimethylsilyl derivative [26].
0379-0738/$ – see front matter ß 2011 Published by Elsevier Ireland Ltd.
doi:10.1016/j.forsciint.2011.06.017
Please cite this article in press as: C.T. Lesar, et al., Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid
(GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique, Forensic Sci. Int. (2011),
doi:10.1016/j.forsciint.2011.06.017
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
G Model
FSI 6488 1–6
e2
C.T. Lesar et al. / Forensic Science International xxx (2011) xxx–xxx
NMR (Nuclear Magnetic Resonance) spectroscopy has been
used to determine the purity of reference drug standards and illicit
forensic drug seizures [27]. 1H and 13C NMR have been used
recently to successfully identify GHB and GBL [28a] and to detect
GHB in human saliva [28b]. The advantage of NMR compared to
other methods is that derivatization is not needed [28]. 1H and 13C
NMR separately give good characteristic signals for both GHB and
GBL. However, if the NMR analysis is performed directly on spiked
beverages without any solvent suppression, 1H spectra are
completely dominated by the large H2O peak and no analyte
investigation is possible (NMR spectra of spiked beverages without
any solvent suppression are available upon request).
In this paper, a fast 1H NMR procedure for the direct analysis of
common alcoholic beverages using a new water suppression
technique called PURGE (Presaturation Utilizing Relaxation
Gradients and Echoes) is proposed. The use of the PURGE method
for the quantitation of GHB and GBL in all beverages studied is also
reported. Quantitative NMR for drug analysis has literature
precedence [30] and recently, the PURGE technique has been
used to identify MDMA in the analysis of urine from 5 cases of
MDMA intoxication [31].
proﬁle of the PURGE sequence was measured using the singlet of
an HDO sample (the results are available upon request for three
different saturation powers). The results show that, while the
excitation proﬁle was relatively uniform, there was considerable
attenuation near the HDO resonance and the range of this
attenuation depended on saturation power. The minimum
saturation power that provided adequate water suppression
was chosen. Using a greater saturation power did not improve
PURGE suppression performance but did result in a wider range of
reduced excitation. In general, it was not necessary to reduce the
residual H2O signal to zero but only so that its intensity was about
10 times as great as other analyte peaks. A saturation power of
W1 = 95 Hz was sufﬁcient. PURGE has the further signiﬁcant
advantage that the only parameters needing adjustment are the
pre-saturation power, relaxation delay and transmitter offset.
This makes it easy to implement even for non spectroscopists. In
practice, little or no adjustment of the presaturation power is
necessary once the experiment has been optimized. The
disadvantages of this method is that labile protons are suppressed
in the spectra. In addition PURGE can only suppress one resonance
at a time.
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
74
2. Materials and methods
75
2.1. Chemicals
3.2. Detection of regions of possible overlap with GHB and GBL signals
in the beverages studied
140
141
The spectra of GHB and GBL in water using the PURGE water
suppression method were ﬁrst recorded and the 1H MNR data (i.e.
chemical shift, peak multiplicity and coupling constants) are
shown in the supplementary material section (supplementary
Tables 1 and 2). All peaks corresponding to the proton signals for
both compounds were clearly identiﬁed. Subsequently, spectra of
all beverages in Table 1 were acquired to identify regions of
possible overlap with GHB and GBL signals. These blank analyses
were all the more important since GBL had previously been
detected as a natural component of wine and in beverages
involving the fermentation of white and red grapes [33]. The
beverages alone were scanned 128 times. Apart from the ethanol
quartet which overlaps with the signal at 3.58 ppm for GHB, the
beverages studied had no peak in the area of interest (i.e. 2.59, 2.30,
4.46 ppm for GBL and 1.77, 2.21 ppm for GHB) except for beer,
white and red wine. These three beverages showed some peaks
between 2.59 and 2.00 ppm and also minute peaks between 1.60
and 1.90 ppm.
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
3.3. Detection of GHB and GBL in spiked beverages
160
NMR spectra of the solutions listed in Table 1 were recorded
at the following concentrations: 0.0025, 0.005, 0.01, 0.025, 0.5,
0.1, 0.25, 0.5, 1.0, 2.5, 5.0% (w/v) for NaGHB and % (v/v) for GBL.
Data collection time differed depending on the concentration of
the analytes, as a better signal to noise ratio is obtained by
increasing the number of scans. The amount of scans needed for
each sample was determined so that in each case, the signal (S)
to noise (N) ratio was above 10 (S/N > 10). Spectra with a drug
concentration of 0.05% (w/v or v/v) and lower were recorded
after 80 scans, those with a drug concentration of 0.1 –0.5% were
recorded after 64 scans, and spectra for beverages with
concentrations of 1% and above were acquired with 16 scans.
This approach was necessary in order to determine the
sensitivity of the method within a certain time frame and to
optimize the amount of time spent using the spectrometer. In a
legal setting, the number of scans should be the same for all
experiments. This would ensure that the exact same conditions
are always used.
Despite the strong ethanol signals, the spectra clearly showed
all the multiplets of GBL allowing its identiﬁcation at concentra-
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
gamma-Hydroxybutyric acid sodium salt (99%) was purchased from Atlantic
Chemicals, Stratton, UK. gamma-Butyrolactone, 3-trimetyhylsilyl 2,20 ,3,30 -tetradeuteropropionic acid (TSP-d4) and deuterium oxide (D2O) were obtained from
Sigma–Aldrich, Pool Rd, UK. A solution of TSP-d4 in deuterium oxide (0.1 mg/ml)
served as internal standard. It was added to a capillary tube which was coaxially
inserted into the NMR tube. The beverages studied were purchased from a local
supermarket. A single bottle of each beverage was used for all experiments. The
label and source of all beverages used are: Samuel Adams Boston Lager (beer);
Gordon’s Dry Gin (unﬂavored gin); Grey Goose (unﬂavored vodka); Bacardi
Superior Rum (unﬂavored rum); Jim Beam White label (whisky, aged 4 years); Cavit
chardonnay 2008 (white wine); Tudor Creek cabernet sauvignon 2008 (red wine);
Canada Dry Tonic Water (tonic water); Ocean Spray Cranberry Juice Cocktail
(cranberry juice); Coca-Cola (coke and diet coke). GHB solutions were prepared at
5% (w/v) (0.5 g of NaGHB in 10 ml of beverage) and GBL solutions at 5% (v/v) (0.5 ml
GBL in 10 ml of beverage). Concentrations were corrected for the sodium salt of GHB
when preparing the solutions. Serial dilutions, using volumetric ﬂasks, were used
for each beverage to yield the desirable concentration range for limit of detection
studies. The alcoholic content of the mixed beverages was set around 10%, which is
slightly above the average value (8.03%) of mixed drinks in college parties [32].
Wine and beer samples were used as supplied. The pH of all of the solutions was
measured using a pH meter because the rate and amount of interconversion
between GHB and GBL is pH dependant. Solutions were analyzed by NMR
spectroscopy immediately so as to limit the effects of interconversion. Solutions of
GBL were also reanalyzed after ﬁve days to monitor the interconversion between
GBL and GHB using the PURGE technique.
101
2.2. NMR spectroscopy
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
1
All data ( H NMR) were collected on a Bruker Avance III 400 MHz NMR
spectrometer (Bruker BioSpin, Billerica, MA) with a 5 mm inverse probe at 300 K at
the Chemistry Department of Columbia University. Proton NMR experiments were
performed using a acquisition time of 2.73 s, a 90 degree pulse width of 7.12 ms, a
relaxation delay of 2.0 s and 32,768 time domain points. Five hundred microlitres of
each sample were introduced into a 5 mm tube with a coaxial capillary tube. The
coaxial capillary tube contained a solution of 3-trimethylsilyl 2,20 ,3,30 -tetradeuteropropionic acid (TSP-d4) in deuterium oxide providing an internal ﬁeld frequency
lock and the internal standard for quantitation experiments. The spectra obtained
from the 1H NMR runs were analyzed using the program Topspin (Bruker). Onedimensional spectra were obtained using the water suppression technique PURGE
described by Simpson et al. to suppress the signal from water [29].
3. Results and discussion
3.1. Optimization of the PURGE water suppression method
One of the advantages of PURGE water suppression is the
reported uniformity of its excitation proﬁle across the entire
proton spectrum. To conﬁrm this uniformity, the actual excitation
Please cite this article in press as: C.T. Lesar, et al., Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid
(GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique, Forensic Sci. Int. (2011),
doi:10.1016/j.forsciint.2011.06.017
G Model
FSI 6488 1–6
C.T. Lesar et al. / Forensic Science International xxx (2011) xxx–xxx
e3
Table 1
Beverages used, alcohol content, Observed Detection Limits (ODL) for 128 scans, and Limit of Quantitation (LOQ).
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
Beverage
Alcohol content (%)
ODL GHB (%, w/v)
LOQ GHB (%, w/v)
ODL GBL (%, v/v)
LOQ GBL (%, v/v)
Water
Ethanol
Vodka–cranberry juice
Rum and coke
Gin–tonic
Whisky–diet coke
White wine
Red wine
Beer
0
10
10
10
10
10
12.5
13.9
4.75
0.003
0.005
0.005
0.005
0.005
0.01
0.01
0.01
0.01
0.95
0.11
0.92
0.23
0.13
0.98
0.24
0.24
0.21
0.003
0.003
0.01
0.005
0.005
0.005
0.01
0.01
0.03
0.28
0.29
0.30
0.41
0.34
0.31
0.43
0.46
0.32
tions above the ODL (Observed Detection Limit for 128 scans;
deﬁned by signal (S) to noise (N) ratio above 10) and in all the
beverages studied. For GHB, one methylene group (3.58 ppm) is
hidden by the ethanol quartet at concentration below 1%. However,
GHB is still identiﬁable in all beverages at concentrations above the
ODL by the signals at 2.47 and 1.86 ppm. Spectra of the spiked
beverages listed in Table 1 were recorded and examples of such
spectra (GBL and GHB in rum and coke at 0.1% (v/v or w/v)) are
shown in Fig. 1.
If the average volume of a drink is between 150 and 200 ml and
the common dosage of GHB is between 2.5 and 4 g then the average
concentration of the drug in a spiked beverage would range from 1
to 3% (w/v) [10,11]. This study shows that at such concentrations
NMR spectroscopy is a valid technique to directly identify GHB and
GBL in the spiked beverages studied. In addition, the pH values of
all drinks at the concentrations mentioned above were taken as
soon as the solutions were made and right before the NMR
experiments were run. The pHs of all beverages upon spiking are
available in the supplementary material section (supplementary
Tables 3 and 4).
3.4. Reproducibility and observed limit of detection (ODL)
201
To determine the standard deviation (s), eight independent
samples of each beverage with a drug concentration of 0.05% (w/v)
for GHB or (v/v) for GBL were run. Statistical analysis of the
samples was performed and the relative standard deviation of the
method was calculated in each case. As for the limit of detection
(LOD), the very nature of NMR makes it impossible to determine as
it depends on the number of scans and pulse width used for the
experiment. For most modern analytical methods, the LOD may be
divided into two components, instrumental detection limit (IDL)
and method detection limit (MDL). For NMR spectroscopy, the IDL
is, in theory, unlimited since it depends on the number of scans.
The MDL is deﬁned as the smallest amount of an analyte that can
be reliably detected or differentiated from the background for a
particular matrix (by a speciﬁc method). In this study we have
limited the experimental time to 10 min, allowing a maximum of
128 scans. This experimental time gave the Observed Detection
Limits (i.e. ODL; deﬁned by signal (S) to noise (N) ratio above 10)
shown in Table 1. In this case, the ODL is the equivalent of the
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
Fig. 1. NMR spectra of 0.1% (v/v) GBL and 0.1% (w/v) GHB in rum and coke.
Please cite this article in press as: C.T. Lesar, et al., Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid
(GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique, Forensic Sci. Int. (2011),
doi:10.1016/j.forsciint.2011.06.017
G Model
FSI 6488 1–6
e4
C.T. Lesar et al. / Forensic Science International xxx (2011) xxx–xxx
220
221
222
method detection limit. Limit of Quantitation (LOQ) was determined by the equation: ten times the standard deviation i.e.: 10s.
Data for ODL and LOQ are shown in Table 1.
223
3.5. Quantiﬁcation by proton NMR spectroscopy of GHB and GBL
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
Quantitation was done using peak integrals according to the
following: the ratio [integral of the drug multiplet/integral of the
internal standard singlet] was plotted versus the drug concentration. For the following beverages: 10% ethanol in water, gin and
tonic, rum and coke, whisky and diet coke and vodka and cranberry
juice, calibration curves were obtained for both GHB and GBL. The
integral of the triplet at 2.59 ppm was used for the quantitation
curves of GBL whereas for GHB, the integral of the triplet at
2.21 ppm was used. For beer, quantitation analysis was performed
with GHB only and the integral of the multiplet (apparent quintet)
at 1.77 ppm was used (interferences were present around
2.21 ppm). For both red and white wines, the presence of minute
interferences prevented accurate quantitation.
Spectra for beverages below the ODL (Observed Limit of
Detection) were not used to establish the calibration curves. A
single calibration curve was used to establish the calibration model
and each calibration point was prepared and analyzed 4 times
independently (at 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0% (w/
v) for NaGHB and % (v/v) for GBL). The method produced a linear
response over the range studied with correlation coefﬁcients of
0.99 or greater in all matrices tested. Correlation coefﬁcients less
than 0.998 were due to experimental errors with integrations.
Calibration curves obtained for the following beverages: 10%
ethanol in water, vodka–cranberry juice, gin–tonic, whisky–diet
coke, rum and coke and beer; are available upon request. The
spectra of both drugs in all beverages with the integral curves
placed above the peaks being integrated are available in the
supplementary material section. The spectra are shown at a 0.1%
concentration with the tables of all integral results. Spectra for
other concentrations are available upon request.
These calibration curves prove that at the average concentration of the drug in a spiked beverage (1–3% (w/v or v/v)) the NMR
technique can be used to directly quantify the amount of drugs
present despite the presence of strong ethanol peaks. In the
present study, samples were analyzed with a relaxation delay of
2 s. The integrals of analyte and standard did not strictly reﬂect the
number of moles present but the calibration curve established
their relative responses. For this approach to be valid, an unknown
sample must be run under the exact same conditions (pulse angles,
delays, dummy scans, and saturation power) as the calibration
samples. The calibration curves established in this work can be
utilized to quantitate samples with the same matrices as the ones
we performed our experiments on. If the matrix is different from
the ones we used, a blank sample should be run before establishing
the calibration curve to ensure that there is no interference with
the signals for GHB and GBL.
270
3.6. Bias study: validation of the method for quantitation
271
272
273
274
275
276
277
278
279
280
281
Three samples of known concentration spiked with GHB and
one sample spiked with GBL were prepared. Those samples were
prepared independently from the samples used to establish the
calibration curves. The calibration curves previously established
were used to calculate the concentrations of each beverage by
simply calculating the ratio of integrals of drug analyte to standard
and reading from the curve.
At the higher concentrations, concentrations calculated from
the curves are equal to the real concentrations. At the lower
concentration (0.025%, w/v), a 10% variation between the real and
calculated concentration was observed.
3.7. Determination of the relative amounts of GHB and GBL present
282
The PURGE technique was tested on aged samples spiked with
GBL. The purpose was to verify if the PURGE method allowed for
the determination of the relative amounts of GHB formed from the
conversion of GBL. The samples were analyzed immediately after
they were spiked and reanalyzed after 5 days. The samples pH was
measured after spiking. Samples of 5% (v/v) GBL in water and 5% (v/
v) GBL in rum and coke were aged for ﬁve days in a sealed container
at room temperature in the dark. Interestingly, a small amount of
conversion to GHB could already be observed the day the samples
were made. Five days later, the GBL and GHB peaks that were
present were integrated and the amount of hydrolysis of GBL to
GHB was calculated. For water aged ﬁve days (pH 6.87), the relative
proportion of GHB formed from GBL was 5.2%. For GBL in rum and
coke, (pH 2.79), the relative proportion of GHB formed was 20%. We
observed a slow conversion of GBL into GHB for the water solution
(pH 6.87) and a faster conversion for the rum and coke solution (pH
2.79). This study is consistent with the studies done by Ciolino et al.
in 2001 [13a]. GHB will form in beverages spiked with GBL at
varying rates dependant, among other factors, on pH, until the
system reaches equilibrium. The PURGE technique can be used as
an efﬁcient tool to determine the relative concentration of both
drugs in different matrices, one of them containing ethanol.
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
3.8. Example of forensic application: identiﬁcation of the source of the
GHB present in solution
305
306
For samples directly spiked with NaGHB, the GHB signal for the
methylene protons adjacent to the carbonyl carbon (signal nb 1,
Fig. 2) is pH dependent. In this study, we observed movement of
the chemical shift for signal nb 1 in accordance with DeFrancesco’s
ﬁnding [21]. This shift is due to the conversion of the carboxylate
from NaGHB (A) to the free acid form of GHB (HA) shown in Fig. 2
[21]: The chemical shift for signal 1 in the salt form (A) is
2.22 ppm; for the free acid form (HA); it is 2.46 ppm. The proton
exchange between the two forms happens so rapidly that the NMR
technique cannot distinguish between the two, which is why there
is only one signal with a chemical shift between 2.22 ppm and
2.45 ppm for both forms. At pH 3.3, the free acid (HA)
predominates, whereas at pH 6.1 the carboxylate (A) predominates. At the midpoint of this range at pH 4.7 there is equimolar
distribution of HA and A [21].
In aged samples spiked with GBL, the chemical shift for signal 1
of the GHB formed was observed between 2.44 ppm and 2.45 ppm.
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
Fig. 2. Interconversion between GHB and GBL (top) and interconversion between
GHB free acid and GHB carboxylate (bottom). Chemical shifts for the signal 1 of GHB
are indicated in parenthesis.
Please cite this article in press as: C.T. Lesar, et al., Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid
(GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique, Forensic Sci. Int. (2011),
doi:10.1016/j.forsciint.2011.06.017
G Model
FSI 6488 1–6
C.T. Lesar et al. / Forensic Science International xxx (2011) xxx–xxx
e5
Table 2
pH of beverages and chemical shift for signal 1 of GHB at 0.1% (w/v) of NaGHB.
Beverage
Alcohol content (%)
pH of beverage
before adding NaGHB
pH of beverage after
adding NaGHB
Shift of GHB
signal 1 (ppm)
Vodka–cranberry juice
Gin–tonic
White wine
Red wine
Whisky–diet coke
Beer
Rum and coke
10% ethanol
Water
10
10
12.5
13.9
10
4.75
10
10
0
2.82
2.72
3.50
3.75
3.28
4.45
2.79
6.21
6.87
3.35
3.34
3.80
3.79
4.75
4.92
4.72
9.80
9.15
2.44
2.44
2.46
2.46
2.36
2.32
2.31
2.25
2.25
Table 3
pH of rum and coke and chemical shift for signal 1 of GHB for solutions with 0.1% (w/v) of NaGHB or 0.1% (v/v) of GBL.
pH with NaGHB (0.1%, w/v) and shift of GHB signal 1
pH with GBL (0.1%, v/v) and shift of GHB signal 1
pH 4.72, d 2.31
pH 2.78, d 2.44
pH of rum and coke
pH 2.79
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
According to DeFrancesco’s study, this chemical shift is observed in
solutions where the free acid (HA) predominates [21]. When
beverages were spiked with NaGHB, the chemical shift of the
methylene signal 1 was between 2.22 and 2.46 ppm (see Table 2)
reﬂecting the equilibrium molar ratio of the GHB free acid (HA) and
carboxylate (A) forms [34].
The case of rum and coke (see Table 3) is an interesting example
of how the PURGE method can be used for forensic application. In
this case, the source of the GHB from an aged sample can be
determined (as long as the equilibrium is not reached) from the
shift of signal 1using the PURGE method directly on the spiked
alcoholic beverage. If the source of GHB is GBL, the chemical shift
for signal 1 of GHB is 2.44 ppm (0.1% (w/v), pH 2.78 after
adulteration), if the source is NaGHB, the chemical shift is
2.31 ppm for signal 1 (pH 4.72 after adulteration). This has
forensic relevance because in some cases, it would allow the
determination of the origin of the GHB present in a seized sample
without any change to the matrix.
Acknowledgments
The authors would also like to thank Colin Deppen and Andrea
Placke for proofreading the manuscript.
345
Appendix A. Supplementary data
346
347
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.forsciint.2011.06.017.
348
References
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
[1] R.H. Roth, N.J. Giarman, Natural occurrences of gamma-hydroxybutyrate in
mammalian brain, Biochem. Pharmacol. 19 (1970) 1087–1093.
[2] H. Laborit, Sodium 4-hydroxybutyrate, Int. J. Neuropharmacol. 3 (1964) 433–452.
[3] H.R. Sumnall, K. Woolfall, S. Edwards, J.C. Cole, Use, function, and subjective
experiences of gamma-hydroxybutyrate (GHB), Drug Alcohol Depend. 92 (2008)
286–290.
[4] V.R. Sanguineti, A. Angelo, M.R. Frank, GHB: a home brew, Am. J. Drug Alcohol
Abuse 23 (1997) 637–742.
[5] Substance Abuse and Mental Health Services Administration (SAMHSA), Ofﬁce of
Applied Studies, gamma-hydroxybutyrate (GHB) abuse in the United States,
Available at: http://www.oas.samhsa.gov/treatan/treana14.htm (last accessed
April 2011).
[6] U.S. Department of Justice, Drug Enforcement Administration, Ofﬁce of Diversion
Control, gamma-hydroxybutyric acid(GHB), Available at: http://www.deadiversion.
usdoj.gov/drugs_concern/ghb/ghb.htm (last accessed April 2011).
[7] Drug Abuse Warning Network. The DAWN Report, Club Drugs 2001 Update,
364
365
Available at: http://dawninfo.samhsa.gov/old_dawn/pubs_94_02 (last accessed
366
March 2010).
367
[8] P. Kam, F. Yoong, gamma-Hydroxybutyric acid: an emerging recreational drug,
368
Anaesthesia 53 (1998) 1195–1198.
369
[9] W. Dean, J. Morgenthaler, S. Fowkes, GHB – The Natural Mood Enhancer, Smart
370
Publications, Petaluma, CA, 1998.
371
[10] European Monitoring Center for Drugs and Drug Addiction, Thematic papers, GHB
372
and its precursor GBL: an emerging trend case study, Available at: www.emcd373
da.europa.eu/attachements.cfm/att_58668_EN_TP_GHB%20and%20GBL.pdf
374
common dosage for GHB spiked beverages (last accessed October 2010).
375
[11] G.P. Galloway, S.L. Frederick-Osborne, R. Seymour, S.E. Contini, D.E. Smith, Abuse and
376
therapeutic potential of gamma-hydroxybutyric acid, Alcohol 20 (2000) 263–269.
377
[12] F. Poldrugo, G. Addolorato, The role of gamma-hydroxybutyric acid in the treat378
ment of alcoholism: from animal to clinical studies, Alcohol 34 (1999) 15–24.
379
[13] (a) L.A. Ciolino, M.Z. Mesmer, R.D. Satzger, A.C. Machal, H.A. Mccauley, A.S.
380
Mohrhaus, The chemical interconversion of GHB and GBL: forensic issues and
381
implications, J. Forensic Sci. 46 (2001) 1315–1323;
382
(b) F.A. Long, F.B. Dunkle, W.F. McDevitt, Salt effects on the acid-catalyzed
383
hydrolysis of gamma-butyrolactone. II. Kinetics and the reaction mechanism, J.
384
Phys. Colloid Chem. 55 (1951) 829–842;
385
(c) F.A. Long, L. Friedman, Determination of the mechanism of gamma-lactone
386
hydrolysis by a mass spectrometric method, J. Am. Chem. Soc. 72 (1950) 3692–3695;
387
(d) J.S. Chappell, The non-equilibrium aqueous solution chemistry of gamma-hydro388
xybutyric acid, JCLIC 12 (2002) 20–27.
389
[14] D.M. Wood, C. Warren-Gash, T. Ashraf, S.L. Greene, Z. Shalther, C. Trivedy, S.
390
Clarke, J. Ramsey, D.W. Holt, P.I. Dargan, Medical and legal confusion surrounding
391
gamma-hydroxybutyrate (GHB) and its precursors gamma-butyrolactone (GBL)
392
and 1,4-butandiol (1,4 BD), QJM 101 (2008) 23–29.
393
[15] S.A. Hennessy, S.M. Moane, S.D. McDermott, The reactivity of gamma-hydroxy394
butyric acid (GHB) and gamma-butyrolactone (GBL) in alcoholic solutions, J.
395
Forensic Sci. 49 (2004) 1220–1229.
[16] Addition of gamma-hydroxybutyric acid to Schedule. 1, Fed. Regist. 65 (2000) Q2396
397
13235–13238.
398
[17] Controlled Substances Act. 21 USC 802 (32) 1970.
399
[18] United Nations Ofﬁce for Drug Control and Crime Prevention (ODCCP), Substances
400
placed under treaty control, in: ODCCP (Ed.), ODCCP Update, ODCCP, Vienna,
401
2001, p. 14.
402
[19] European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), Report on
403
the risk assessment of GHB in the framework of the joint action on new synthetic
404
drugs, Luxembourg: Ofﬁce for Ofﬁcial Publications of the European Communities,
405
2002, pp. 17–18. Available at: www.emcdda.europa.eu/attachements.cfm/
406
att_1147_EN_ghb_ﬁnal_riskassessment_report.pdf (last accessed October 2010).
407
[20] V.L. Brewster, H.G. Edwards, M.D. Hargreaves, T. Munshi, Identiﬁcation of date408
rape drug GHB and its precursor GBL by Raman spectroscopy, Drug Test Anal. 1
409
(2009) 25–31.
410
[21] J.V. DeFrancesco, M.R. Witkowski, L.A. Ciolino, GHB free acid. I. Solution formation
1
411
studies and spectroscopic characterization by H NMR and FT-IR, J. Forensic Sci.
412
51 (2006) 321–329.
413
[22] J.S. Chappell, A.W. Meyn, K.K. Ngim, The extraction and infrared identiﬁcation of
414
gamma-hydroxybutyric acid (GHB) from aqueous solutions, J. Forensic Sci. 49
415
(2004) 52–57.
416
[23] M.Z. Mesmer, R.D. Satzger, Determination of gamma-hydroxybutyrate (GHB) and
417
gamma-butyrolactone (GBL) by HPLC/UV-VIS spectrophotometry and HPLC/ther418
mospray mass spectrometry, J. Forensic Sci. 43 (1998) 489–492.
419
[24] K.M. Andera, H.K. Evans, C.M. Wojcik, Microchemical identiﬁcation of gamma420
hydroxybutyrate (GHB), J. Forensic Sci. 45 (2000) 665–668.
Please cite this article in press as: C.T. Lesar, et al., Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid
(GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique, Forensic Sci. Int. (2011),
doi:10.1016/j.forsciint.2011.06.017
G Model
FSI 6488 1–6
e6
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
C.T. Lesar et al. / Forensic Science International xxx (2011) xxx–xxx
[25] E. Kaufmann, A. Alt, Determination of GHB in urine and serum by LC/MS using a
simple one-step derivative, Forensic Sci. Int. 168 (2007) 133–137.
[26] F.J. Couper, B.K. Logan, Determination of gamma-hydroxybutyrate (GHB) in
biological specimens by gas chromatography–mass spectrometry, J. Anal. Toxicol.
24 (2000) 1–7.
[27] P.A. Hays, Proton Nuclear Magnetic Resonance spectroscopy (NMR) methods for
determining the purity of reference drug standards and illicit forensic drug
seizures, J. Forensic Sci. 50 (2005) 1342–1360.
[28] (a) S.L. Chew, J.A. Meyers, Identiﬁcation and quantitation of gamma-hydroxybutyrate (NaGHB) by nuclear magnetic resonance spectroscopy, J. Forensic Sci. 48
(2003) 292–298;
(b) M. Grootveld, D. Algeo, C.J. Silwood, J.C. Blackburn, A.D. Clark, Determination
of the illicit drug gamma-hydroxybutyrate (GHB) in human saliva and beverages
by 1H NMR analysis, Biofactors 27 (2006) 121–136.
[29] A.J. Simpson, S. Brown, Purge NMR: effective and easy solvent suppression, J.
Magn. Reson. 175 (2005) 340–346.
[30] G.F. Pauli, B.U. Jaki, D.C. Lankin, Quantitative 1H NMR: development and
potential of a method for natural products analysis, J. Nat. Prod. 68 (2005)
133–149.
[31] J. Liu, J. Decatur, G. Proni, E. Champeil, Identiﬁcation and quantitation of 3,4methylenedioxy-N-methylamphetamine (MDMA, ecstasy) in human urine by 1H
NMR spectroscopy. Application to ﬁve cases of intoxication, Forensic Sci. Int. 194
(2010) 103–107.
[32] N.P. Barnett, J. Wei, C. Czachowski, Measured alcohol content in college party
mixed drinks, Psychol. Addict. Behav. 23 (2009) 152–156.
[33] (a) J. Vose, T. Tighe, M. Schwartz, E. Buel, Detection of gamma-butyrolactone
(GBL) as a natural component in wine, J. Forensic Sci. 46 (2001) 1164–1167;
(b) S. Elliott, V. Burgess, The presence of gamma-hydroxybutyric acid (GHB) and
gamma-butyrolactone (GBL) in alcoholic and non-alcoholic beverages, Forensic
Sci. Int. 151 (2005) 289–292.
[34] J. Buckingham, F. Macdonald, Dictionary of Organic Compounds, sixth ed.,
Chapman & Hall, New York, 1995.
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
Please cite this article in press as: C.T. Lesar, et al., Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid
(GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique, Forensic Sci. Int. (2011),
doi:10.1016/j.forsciint.2011.06.017

Download Report