Chapter 3

The Analysis of LSD

Learning Objectives
To have an appreciation of LSD as a controlled substance.
To be aware of the sampling procedures and descriptions required for
LSD analysis.
To understand the extraction of LSD from blotter acids for analysis.
To be aware of the chemical and uorescence testing procedures for LSD.
To understand the principles of TLC analysis of LSD.
To be aware of the conrmatory techniques available for the analysis
of LSD.
3.1 Introduction
While a large number of drugs are known which are of plant origin, or have plant
products as starting materials for the synthesis of the drugs, there are, equally, a
number of drugs of fungal origin. Of these, perhaps lysergic acid diethylamide
(LSD) (1) is the most famous, i.e. well known, and it is on this drug that
this chapter focuses. Interestingly, the drug is an indole alkaloid which presents
special difculties and opportunities in terms of drugs analysis.
Lysergic acid diethylamide (LSD) is one of the most potent hallucinogens
known to man. It was rst synthesized in 1938 and was discovered to be psy-
choactive in 1943. It was initially used, experimentally, in the treatment of mental
disorders but has not been used in this way for some 30 years. LSD encountered
in the illicit drugs market of today is produced in clandestine laboratories. These
are rarely detected because they make a large quantity of LSD, which lasts for an
extremely long period of time, since only very small doses are administered and
subsequent syntheses are not required [1]. LSD is, in the main, prepared from
The Analysis of Controlled Substances. Michael D. Cole
Copyright 2003 John Wiley & Sons, Ltd.
ISBNs: 0-471-49252-3 (HB); 0-471-49253-1 (PB)
38 Analysis of Controlled Substances
HN
N
H
CH
3
CON(C
2
H
5
)
2
1
lysergic acid, via a series of complex reactions which require careful monitor-
ing and control. The forensic scientist will see the resulting drug in a number
of differing dosage forms. The materials may be added to inert substrates or to
sugar cubes, or mixed with molten gelatin which is then cooled and cut into
small pieces containing the appropriate dose. These latter are known as window
panes. However, these dose forms suffer from great inhomogeneity and the vast
majority of LSD observed in the forensic science laboratory today is encountered
in the form of blotter acid. In this form, an absorbant paper is dipped into a
solution of LSD, and then dried. Such a procedure allows an even distribution
of the drug through the paper. A typical blotter acid dose contains between 30
and 150 g of LSD per dose. Blotter papers are frequently decorated, with some
examples being shown on Plates 3.1 and 3.2, and represent the dose form on
which our discussion will centre.
LSD doses vary between users and with the desired effect. Commonly, the
doses lie in the range between 30 and 150 g of LSD per dose, although stronger
doses of 150 to 400 g of LSD per dose (or even higher) are sometimes encoun-
tered. The onset of effect takes between 30 and 120 min and lasts between 6 and
14 h. After-effects can last for up to 24 h.
In terms of legislative control, in the United Kingdom lysergamide, lysergide
and any N-alkyl derivatives of lysergamide are controlled as Class A drugs. In
the United States, LSD is controlled as a Schedule I drug. As part of the forensic
science process, therefore, it is necessary to prove the presence of the drug in
any sample thought to contain LSD.
3.2 Qualitative Identication of LSD
The process of analysis of LSD blotter acid follows the same general principles
and sequence as for other controlled substances, namely physical description,
presumptive testing, TLC and conrmatory analysis. These processes are dis-
cussed below.
The Analysis of LSD 39
Plate 3.1 Bird of paradise pattern on LSD blotter acid, covering the whole sheet of the
paper. Copyright Michael D. Cole, Anglia Polytechnic University, Cambridge, UK, and
reproduced with permission.
3.2.1 Sampling and Physical Description of LSD Blotter Acid
As with all forensic science analyses, the rst stage in the process is a full
physical description of the material under investigation. In the case of blotter
acids, this includes a count of the number of dose units, the size of each of the
dose units (length breadth), whether they t together, the number and depth of
40 Analysis of Controlled Substances
Plate 3.2 Illustration of a ghost on LSD blotter acid, with each image covering a few
dose units. Copyright Michael D. Cole, Anglia Polytechnic University, Cambridge, UK,
and reproduced with permission.
the perforations of the dose units and a note of the pattern and whether it covers
all, some, or single dose units (see Plates 3.1 and 3.2).
DQ 3.1
Why is it necessary to record in such detail the physical information
relating to an LSD seizure?
Answer
By recording such information, it may be possible to relate one or more
samples to each other. This may be especially important when seizures
from different occasions are being compared, or comparison between
seizures is being made in different laboratories.
Having carried out a full physical description of the seizure, items must be chosen
for analysis. While it might be assumed that the dose units are all identical,
The Analysis of LSD 41
this might not be the case and it is therefore necessary to sample a number of
them from the seizure. The United Nations Drug Control Programme (UNDCP)
recommends the following procedure. For sample sizes up to 10 dose units, all
should be analysed, while for sample sizes between 11 and 27 dose units, three
quarters of the items should be selected at random (the number being rounded
up to the next highest integer). For sample sizes in excess of 28 dose units, 50%
should be selected at random, with a minimum of 21 dose units up to a maximum
of 50 units [2].
DQ 3.2
How might random samples be chosen?
Answer
A random sample means that every item in a population has an equal
chance of being chosen. Simply choosing materials by eye does not
satisfy this criterion. Each of the dose units should be assigned a number,
starting at 1 and ending with the last number (i.e. the number of items in
the sample). The materials to be chosen should then be picked by using
either a computerized random-number generator or random-number
tables. Whichever method is used, it should be documented.
3.2.2 Extraction of LSD Prior to Analysis
Since the drug is impregnated onto a paper substrate, it is necessary to extract
the material prior to analysis. In order to do this for presumptive testing or qual-
itative analysis, the extraction can simply be achieved by mixing the test sample
for 30 s with sufcient methanol to achieve a sample concentration of 1 mg
LSD ml
1
[2]. Alternatively, a methanol/water (1:1) mixture has been reported
to extract the LSD more efciently [3, 4]. It should be remembered that any
solid material should be removed from the extract prior to any chromatographic
analysis being carried out. This can be achieved either by centrifugation or by
passing the extract through a 5 m lter.
SAQ 3.1
Why should the solid material be removed prior to analysis?
If quantitative analyses are to be carried out, it is necessary to completely extract
the LSD from the paper. This can be achieved by suspending the material in a
large volume (15 ml is suggested) of 1% tartaric acid solution in a separatory
funnel. The mixture is extracted three times with an equal volume of chloroform
and then the aqueous layer is basied with 1 M NaHCO
3
. The resulting mixture
should be extracted, three times, with an equal volume of chloroform, and the
42 Analysis of Controlled Substances
chloroform extracts combined, ltered or centrifuged, and evaporated under a
stream of nitrogen. The residue should then be reconstituted in a known volume
of solvent [2]. Other acids have been used (summarized in Veress [3]), although
the physico-chemical principles remain the same.
SAQ 3.2
What are the physico-chemical principles behind using an acid to extract the LSD
from the paper and the subsequent processing?
3.2.3 Presumptive Testing for LSD
The presumptive tests for LSD involve a uorescence test and a chemical test.
3.2.3.1 Fluorescence Testing for LSD
One of the properties of LSD that can be exploited during the identication
process is its uorescence. In such a test, the original dosage form, or a drop of
the methanolic extract from the dose form, is placed on a lter paper and allowed
to dry. The material is then observed under long-wavelength UV light (360 nm).
If LSD is present, blue uorescence will be observed.
DQ 3.3
If a methanol extract solution is used, what are the appropriate positive
and negative controls and what do they show?
Answer
If methanol is used, the appropriate negative control is methanol alone
(not exposed to sample or drug). The relevant positive control is a
methanolic solution of LSD at approximately the same concentration as
the sample provides.
The negative control demonstrates that the uorescence, if observed, is due to
the drug extracted into the methanol. The positive control provides a reference
colour reaction and gives an idea of the intensity of the uorescence that might
be observed.
3.2.3.2 Chemical Testing for LSD
The classical presumptive test for LSD is the Ehrlichs reagent test. In this, 1 g of
the reagent (p-dimethylaminobenzaldehyde) is dissolved in 10 ml of orthophos-
phoric acid. A small amount of the reagent solution is then added either directly
to the test substrate or to the extract to be tested and any colour change observed.
The Analysis of LSD 43
If a blue/purple colour develops, then the presence of LSD may be suspected and
conrmatory tests should be carried out.
DQ 3.4
Why is a conrmatory test required?
Answer
Ehrlichs reagent reacts with a wide range of controlled substances and
other indole alkaloids. A positive reaction does not therefore prove the
presence of a specic drug. This is why it is necessary to carry out an
additional conrmatory test.
3.2.4 Thin Layer Chromatography of Samples Containing LSD
Following the possible identication of the presence of LSD, the next stage in
the analysis is the use of thin layer chromatography (TLC). This is employed
because although it cannot be used to prove the identity of LSD, it can be used as
a rapid, cost-effective method to eliminate those samples which gave a positive
colour reaction in the presumptive tests but which do not contain this drug. These
will be rarer when blotter acids are suspected, but may be more common where
other substrates have been used as the carrier medium for the LSD itself.
The extracts are prepared as described above (see Section 3.2.2). Activated
silica gel plates containing a uorescent dye (which uoresces at 254 nm) are
used. The materials to be tested, plus positive and negative controls, are spotted
onto the plate and the chromatograms developed in the normal way. The solvent
systems which can be used include chloroform/methanol (9:1, by volume) and
chloroform/acetone (1:4, by volume). Following chromatogram development, the
plates are removed from the chromatographic tank, the solvent fronts marked,
and the plates air-dried and observed under short- (254 nm) and long- (360 nm)
wavelength UV light. Under the former lighting conditions, LSD will appear
as a dark spot on a light background, while under the latter conditions it will
appear as a bright spot on a dark background. The chromatogram should then be
developed with Ehrlichs reagent, with which indole alkaloids (including LSD)
react to give a purple product. If a product gives the same results (retardation
factor, R
f
, and colour reaction) as LSD under all of the conditions described,
then further conrmatory tests should be carried out. However, if the materials
do not yield the same physico-chemical responses as LSD under all conditions,
then an exclusion has been achieved.
3.2.5 Conrmatory Tests for the Presence of LSD
Since there are a large number of materials which will produce the same result
as LSD in one or more of the screening tests, it is necessary to conrm the pres-
ence of this drug by using an instrumental technique. Of the methods available,
44 Analysis of Controlled Substances
HPLC with uorescence detection is the most commonly used, although GC
techniques have been reported [4, 5]. HPLC is also used as the means of quan-
tifying LSD.
3.2.5.1 HPLC Analysis of LSD
A number of HPLC methods are available for the analysis of LSD. Two are
presented here, namely (i) an ion-suppression technique, and (ii) an ion-paired
method. The majority of techniques that are used exploit the same physico-
chemical characteristics of the systems as those described here.
The rst method is a reversed-phase method, using the HPLC system conditions
described in Table 3.1. LSD is particularly amenable to uorescence detection,
which also has the advantage of providing both selectivity and sensitivity.
SAQ 3.3
How is selectivity of detection achieved when using a uorescence detector to
detect analytes in an HPLC eluant?
DQ 3.5
How does the ion-suppression work in this system?
Answer
At pH 8.0, the LSD will be present in the free base form. It will not be
highly charged and hence will exhibit good chromatographic properties.
The second of the methods [3] uses an ion-pairing system, the conditions of
which are given in Table 3.2. This system is not as specic as that employ-
ing uorescence detection, although it is included here to illustrate that other
separation mechanisms can be employed.
SAQ 3.4
Why is this detection system not as specic as the uorescence detection one?
Table 3.1 Typical HPLC (ion-suppression) operating conditions and parameters
used for the identication and quantication of LSD [2]
System/parameter Description/conditions
Column ODS
a
silica: 10 cm 4.6 mm i.d.; 5 m particle size
Solvent Methanol (65%): 25 mM Na
2
HPO
4
, pH 8.0 (35%)
Flow rate 1 ml min
1
Detection Fluorescence: excitation , 320 nm; emission , 400 nm
a
ODS, octadecasilyl.
The Analysis of LSD 45
Table 3.2 Typical HPLC (ion-pairing) operating conditions and parameters used for the
identication and quantication of LSD [3]
System/parameter Description/conditions
Column ODS
a
silica: 25 cm 4.6 mm i.d.; 0.8 m particle size
Solvent Acetonitrile (45%): 50 mM KH
2
PO
4
/5 mM C
8
H
17
SO
3
Na, pH
3.5 (55%)
Flow rate 0.9 ml min
1
Detection wavelength 220 nm
a
ODS, octadecasilyl.
SAQ 3.5
What is the chromatographic mechanism operating in the solvent system des-
cribed in Table 3.2?
3.2.5.2 GC Analysis of LSD
Gas chromatographic analysis of LSD is problematic because of the relatively
limited volatility of this (controlled) substance [3]. However, GC-based methods
have been applied, e.g. GCMS [5]. The conditions reported for such analysis
are shown in Table 3.3.
SAQ 3.6
What are the potential difculties in analysing LSD by using isothermal gas
chromatography?
3.2.5.3 Identication of LSD by Microscope FTIR Spectroscopy

More recently, it has become possible to identify LSD in situ, without the need for
extraction of the drug [6]. In this technique, LSD blotter acid is described (see
Table 3.3 GCMS operating conditions and parameters used for the identica-
tion of LSD [5]
System/parameter Description/conditions
Column BP-5: 25 m 0.2 mm i.d.; d
f
, 0.33 m
Injection temperature 270

C
Column oven temperature
programme
100

C; rising at 24

C min
1
to 270

C;
isothermal for 35 min
Detection Mass spectrometric
Split ratio
a
a
Not reported.

This is a technique used to measure the IR adsorption or reection spectra of very small samples. In this method,
a sample is placed on a KBr disc and a microscope is then used to focus the IR beam onto the material.
46 Analysis of Controlled Substances
Section 3.2.1) and then subjected to a simple extraction procedure, followed by
microscope FTIR spectroscopy directly on the extract. In this approach, the blotter
acid is rst soaked in water for 1 s, which swells the bres of the papers and was
found to facilitate the extraction of the drug for further spectroscopic analysis.
The excess water was then removed from the blotter, which was subsequently
placed on a KBr disc and heated to 120

C for 1 min. Dichloromethane/ammonia

(100:1) was added to the blotter, which dissolved the LSD. The solution was
then removed by using a microsyringe, placed on a KBr disc, and its spectrum
recorded.
DQ 3.6
Why is ammonia added to the dichloromethane?
Answer
LSD is a basic drug. The ammonia will convert any LSD from the salt
form to the free base state. In this form, the LSD is more soluble in organic
solvents such as dichloromethane and thus the extraction process will be
more efcient.
The IR spectrum was obtained by scanning in the wavenumber range 700
4000 cm
1
, with 30 scans at a resolution of 4 cm
1
being collected. From such
data it was possible to identify LSD directly.
SAQ 3.7
Why were 30 scans taken and what is the advantage of using the scan range from
700 to 4000 cm
1
?
Summary
LSD is one of the most potent hallucinogens known to man. This drug can occur
in the form of either tiny tablets or as gelatin blocks, but is most commonly found
as blotter paper small pieces of highly decorated paper into which the LSD
has been impregnated. The analysis of blotter acids includes a physical description
of the item, followed by drug extraction from the paper. The extract is then used
for uorescence and colour tests to determine if LSD may be present. Following
this, TLC and HPLC with uorescence detection can be used to conrm the
presence of the LSD, although GC methods are available for such analysis. An
interesting new technique, using microscope FTIR spectroscopic analysis of the
drug in situ on the paper, has also been developed.
The Analysis of LSD 47
References
1. Huizer, H., personal communication.
2. United Nations Drug Control Programme, Recommended Methods for Testing Lysergide (LSD),
Manual for use by national narcotics laboratories, United Nations Division of Narcotic Drugs,
New York, 1989.
3. Veress, T., Study of the extraction of LSD from illicit blotters for HPLC determination, J.
Forensic Sci., 38, 11051110 (1993).
4. Ripani, L., Schiavone, S. and Garofano, L., GC Quantitative determination of illicit LSD, J.
Forensic Sci., 39, 512517 (1994).
5. Kilmer, S. D., The isolation and identication of lysergic acid diethylamide (LSD) from sugar
cubes and a liquid substrate, J. Forensic Sci., 39, 860862 (1994).
6. Hida, M. and Mitsui, T., Rapid identication of lysergic acid diethylamide in blotter paper by
microscope FT-IR, Anal. Sci., 15, 289291 (1999).
Plate 3.1MBird of paradise pattern on LSD blotter acid, covering the whole sheet of the paper.
Copyright Michael D. Cole, Anglia Polytechnic University, Cambridge, UK, and reproduced
with permission.
Plate 3.2MIllustration of a ghost on LSD blotter acid, with each image covering a few dose
units. Copyright Michael D. Cole, Anglia Polytechnic University, Cambridge, UK, and
reproduced with permission.