New Synthesis and Characterization of (+)-Lysergic Acid
Diethylamide (LSD) Derivatives and the Development of a
Microparticle-Based Immunoassay for the Detection of LSD and Its
Metabolites
Zhuyin Li, K. Goc-Szkutnicka, A. J. McNally,* I. Pilcher, S. Polakowski, S. Vitone, Robert S. Wu, and
S. J. Salamone
Drug Monitoring, Diagnostics Research and Product Development, Roche Diagnostic System, Inc., 1080 U.S.
Highway 202, Somerville, New Jersey 08876-3771. Received April 24, 1997X
In this paper are reported the synthesis and characterization of three LSD derivatives. On the basis
of several analytical characterization studies, the most stable derivative has been selected and a
procedure to covalently link the derivative to polystyrene microparticles through a carrier protein
has been developed. In addition, two new LSD immunogens have been synthesized and characterized,
and from these immunogens antibodies that recognize not only LSD but also several major LSD
metabolites have been generated. Using the selected derivative and antibody, a homogeneous
microparticle-based immunoassay has been developed for the detection of LSD in human urine with
the required sensitivity and specificity for an effective screening assay. The performance of this LSD
OnLine assay has been evaluated using the criteria of precision, cross-reactivity, correlation to the
Abuscreen LSD RIA and GC/MS/MS, assay specificity, and limit of detection.
INTRODUCTION
(+)-Lysergic acid diethylamide (LSD,1 Scheme 1) is a
highly potent drug that acts on the central nervous
system and alters sensory perception, states of consciousness,
and thought processes. By causing these altered
states, the drug produces severe visual and auditory
hallucinations. In addition to these physical effects, the
use of LSD has been and continues to be a problem for
drug and law enforcement agencies around the world (1-
4). Making the problem even more complex is the fact
that the detection of LSD in body fluids of users is
difficult because the quantities typically ingested are very
small (100-250 ?g/dose) (4). To date, limited research
has been conducted on the chemical properties of LSD.
It is, however, known that LSD has an inherent fluorescence,
which can be excited at 320 nm and emits at 445
nm. Additionally, under UV light irradiation, LSD can
undergo catalytic hydration at the C-9,10 double-bond
position. Once hydration occurs, the loss of fluorescence
at 445 nm is observed. In alkaline solution, LSD undergoes
an epimerization at the C-8 position, resulting in
partial formation of iso-LSD. LSD is also unstable under
prolonged heat exposure, but the mechanism of the
thermal decomposition is not yet fully understood (5, 6).
Under physiological conditions, LSD is rapidly and
extensively converted to several known and unknown
metabolic products. At present, several metabolites of
LSD in the human body, such as N-demethyl-LSD (nor-
LSD), 2-oxo-3-hydroxy-LSD, 13-hydroxy-LSD, and 14-
hydroxy-LSD, have been tentatively identified; yet only
one metabolite (nor-LSD) and the parent compound, both
excreted at 1% of the total dose, have been conclusively
identified (6-10).
Currently, the measurements of LSD and its metabolites
in biological fluids rely on radioimmunoassay methods
or HPLC fluorescence methods and very specialized
GC/MS/MS methods for confirmation (7-13). These
methods produce undesirable radioactive waste or require
extensive pretreatment of samples, very specialized
equipment, and highly trained personnel. There has
been much interest in recent years, because of the
reported increased abuse, to develop nonisotopic, highly
automated, homogeneous analytical methods to detect or
screen for LSD abuse. Due to the lower sensitivities of
nonisotopic immunoassays, the instability of LSD under
both physiological and nonphysiological conditions, and
the lack of information about the majority of the metabolites,
the development of nonisotopic immunoassays
to date has been a challenge. It is, therefore, extremely
important to systematically synthesize LSD derivatives
and study the stability of these derivatives for the
development of a nonisotopic immunoassay. A conjugate
procedure for making a LSD microparticle is also critical
for obtaining a highly sensitive LSD assay. In addition,
it is very important to generate antibodies that are
capable of recognizing not only LSD but also its major
metabolites. These antibodies must have low cross-
* Author to whom correspondence should be addressed.
X Abstract published in Advance ACS Abstracts, October 15,
1997.
1 Abbreviations: BTG, bovine thyroglobulin; DMSO, dimethyl
sulfoxide; DMF, N,N-dimethylformamide; CMC, N-cyclohexyl-
N¢-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate;
EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked
immunosorbent assay; GC/MS/MS, gas chromatography/tandem
mass spectrometry; KPi, potassium phosphate; NHB??H2O, Nhydroxybenzotriazole
hydrate; NHS, N-hydroxysuccinimide;
LSD, (+)-lysergic acid diethylamide; nor-LSD, N-demethyl-LSD;
NMR, nuclear magnetic resonance; SAMHSA, substance abuse
and mental health services administration; TEA, triethylamine;
THF, tetrahydrofuran; TLC, thin-layer chromatography; TNBS,
trinitrobenzenesulfonic acid.
Scheme 1. Structure of LSD
896 Bioconjugate Chem. 1997, 8, 896-905
S1043-1802(97)00059-1 CCC: $14.00 © 1997 American Chemical Society
reactivity to structurally related compounds that are not
substances of abuse.
In this paper, we describe the synthesis and characterization
of new LSD derivatives used in the development
of a microparticle-based OnLine immunoassay. We
also describe a new procedure to make a LSD microparticle.
Like other OnLine immunoassays, this assay
is based on the principle of the kinetic interaction of
microparticles in solution (KIMS) (14), in which the drug
content in the urine is directly proportional to the
inhibition of the microparticle agglutination. Four LSD
derivatives were synthesized, two of which (3 and 7,
Scheme 3) were used to prepare immunogens (4 and
8, Scheme 3) for antibody production and three of
which (6, 7, and 10, Schemes 2 and 3) were examined
for best stability. From this work the most stable
derivative was selected for preparing the conjugate. For
the development of this microparticle-based assay, the
selected LSD derivative was covalently coupled to a
carrier protein, and this conjugate was then covalently
linked to microparticles. These newly developed LSD
microparticles, together with the properly selected antibodies,
were then developed into a competitive displacement
immunoassay for LSD with a detection limit of 0.2
ng/mL LSD.
MATERIALS AND METHODS
Reagents. All solvents were obtained from Fisher
Scientific (Pittsburgh, PA) unless specified. All flash
grade silica gel and silica gel preparative TLC plates were
obtained from E. M. Science (Gibbstown, NJ). Protein
concentrations were determined by using the Bradford
protein assay reagents (15) purchased from Bio-Rad
(Hercules, CA), and 2-oxo-3-hydroxy-LSD was purchased
from Radian (Austin, TX). LSD, nor-LSD, 1-(3-aminobutyl)-
N,N-diethyl-D-lysergamide, and 1-(3-aminopropyl)-
N,N-diethyl-D-lysergamide (1a and 1b, Scheme 2) (16)
and Abuscreen RIA were prepared by Roche Diagnostic
Systems (Somerville, NJ). LSD antibodies for the
Abuscreen RIA were generated using a LSD analogue
derivatized through the indole nitrogen and conjugated
to BSA. Carboxylated polystyrene microparticles were
obtained from Bangs Laboratories (Carmel, IN). CMC,
NHB??H2O, ovalbumin, and other reagents were obtained
from Sigma (St. Louis, MO).
Instrumentation. Fluorescence measurements were
carried out by using an LS-5B luminescence spectrometer
(Perkin-Elmer, Norwalk, CT). The excitation wavelength
was set at 320 nm, and the emission wavelength was
measured at 445 nm. Light irradiation was performed
using a 20 W desk-top fluorescent light. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded at
400 MHz on an XL-400 NMR spectrometer (Varian,
Palo Alto, CA); coupling constants are given in hertz (Hz),
and CDCl3 was used as the solvent. The abbreviations
used are as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. The OnLine immunoassay was performed
using an Olympus AU800 automated analyzer (Olympus,
Lake Success, NY).
Synthesis of LSD Labels. Synthesis of 1-(3-Aminobutyl)-
N,N-diethyl-d-lysergamide and 1-(3-Aminopropyl)-
N,N-diethyl-d-lysergamide (2a and 2b, Scheme 2).
A solution of 900 mg (1.7 mmol) of 1a in 25 mL of
methanol was treated with 0.385 mL (12.3 mmol) of
anhydrous hydrazine and stirred at room temperature
overnight. The reaction mixture was concentrated at
reduced pressure. The residue was treated with 25 mL
of a mixture of 9:1 methylene chloride/methanol, and the
insoluble solids were filtered off. The filtrate was chromatographed
on 200 g of silica gel using 25% methanol
in methylene chloride as an eluent to elute front-running
impurities, followed by 2% triethylamine/25% methanol
in methylene chloride as an eluent to elute the product
to yield 563 mg (83%) of 2a as a yellow amorphous solid:
1H NMR (400 MHz, CDCl3) ? 1.17 (3H, t, J ) 7.1), 1.24
(3H, t, J ) 7.1), 1.50-1.60 (2H, m), 1.82-1.92 (2H, m),
2.59 (3H, s), 2.62-2.92 (6H, m), 3.02-3.10 (1H, m), 3.18-
3.26 (1H, m), 3.37-3.57 (6H, m), 3.84-3.92 (1H, m), 4.08
(2H, t, J ) 6.8), 6.33 (1H, s), 6.79 (1H, s), 7.11-7.20 (3H,
m); MS, m/e 394 (M+); HR-EI MS calcd for M+ 394.2733,
found 394.2731. Likewise, 2b was obtained using an
analogous procedure in 80% yield.
Synthesis of 1-[[[(4-Isothiocyanatophenyl)carbonyl]-
amino]butyl]-N,N-diethyl-d-lysergamide (3, Scheme 3). A
solution of 370 mg (0.94 mmol) of 2a in 15 mL of
anhydrous THF was cooled to 0 °C and treated with a
solution of 190 mg (0.96 mmol) of 4-isothiocyanatobenzoyl
chloride (17) in 5 mL of anhydrous THF and then
stirred at 0 °C for 30 min and then at room temperature
overnight; the reaction was driven to completion by
adding 0.14 mL (1.0 mmol) of triethylamine and stirred
at room temperature for 2 h. The reaction mixture was
concentrated at reduced pressure. The residue was
dissolved in methylene chloride, washed with H2O, dried
over anhydrous sodium sulfate, and concentrated at
reduced pressure. The residue was chromatographed on
200 g of silica gel using 3% methanol in methylene
chloride as an eluent to yield 325 mg (62%) of 3 as a tan
amorphous solid: 1H NMR (400 MHz, CDCl3) ? 1.17 (3H,
t, J ) 7), 1.24 (3H, t, J ) 7), 1.55-1.65 (2H, m), 1.80-
2.05 (4H, m), 2.61 (3H, s), 2.63-2.75 (1H, m), 2.85-2.95
(1H, m), 3.04-3.12 (1H, m), 3.18-3.28 (1H, m), 3.35-
3.58 (4H, m), 4.13 (2H, t, J ) 6.4), 5.97-6.03 (1H, m),
6.35 (1H, s), 6.79 (1H, s), 7.10-7.20 (3H, m), 7.24 and
7.67 (4H, AA¢ BB¢q, J ) 8.4); MS, m/e 555 (M+); [R]D)+
47.5° (c 0.91%; CHCl3).
Synthesis of 4¢-[[2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-
[1,1¢-biphenyl]-4-carbonyl Chloride (5, Scheme 2). A
mixture of 2.0 g (8.2 mmol) of 4,4¢-biphenyldicarboxylic
acid in 40 mL of anhydrous THF was treated with 5.0
mL (55.0 mmol) of oxalyl chloride followed by 0.02 mL of
anhydrous DMF. The reaction was stirred at room
temperature for 10 min and then heated to reflux for 90
min. The reaction was then concentrated at reduced
pressure to a yellow oil. This was recrystallized from a
mixture of THF and ether to yield 1.67 g (73%) of the
diacid chloride as yellow needles: 1H NMR (200 MHz,
CDCl3) ? 7.75 and 8.22 (8H, AA¢ BB¢q, J ) 8); MS, m/e
278 (M+).
A solution of 1.67 g (6.0 mmol) of 1,1¢-biphenyl-4,4¢-
dicarbonyl chloride in 65 mL of anhydrous THF was
treated with 710 mg (6.17 mmol) of N-hydroxysuccinimide,
followed by 0.835 mL (6.0 mmol) of triethylamine.
The reaction was stirred at room temperature for 2 h,
after which time it was filtered to remove triethylamine
HCl. The filtrate was concentrated at reduced pressure
to yield 2.0 g (93%) of 5 as a pale yellow solid: IR (CHCl3)
1775, 1742 cm-1; 1H NMR (400 MHz, CDCl3) ? 2.94 (4H,
br s), 7.77 (4H, d, J ) 8.5), 8.23-8.27 (4H, m); MS, m/e
357 (M+).
Synthesis of 1-[3-[[[4¢-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]-
carbonyl][1,1¢-biphenyl]-4-yl]carbonyl]amino]propyl]-N,Ndiethyl-
d-lysergamide (6, Scheme 2). A solution of 850
mg (2.375 mmol) of 5 in 65 mL of anhydrous THF under
argon was cooled to 0 °C in an ice bath and then treated
with a solution of 900 mg (2.365 mmol) of 2b in 50 mL of
anhydrous THF and 0.6 mL (4.3 mmol) of triethylamine
added dropwise over a 20 min period. The reaction
mixture was stirred at 0 °C for 1 h and then warmed to
room temperature, with stirring, for 1 h. The mixture
LSD Derivatives Bioconjugate Chem., Vol. 8, No. 6, 1997 897
was concentrated at reduced pressure, and the residue
was dissolved in 100 mL of methylene chloride. The
solution was washed with 100 mL of H2O, 100 mL of
saturated sodium bicarbonate solution, and 100 mL of
saturated brine solution, dried over anhydrous sodium
sulfate, and concentrated at reduced pressure to a brown
residue. This was chromatographed on a short column
of 100 g of silica gel using first methylene chloride as an
eluent, then 9:1 methylene chloride/ether as an eluent
to elute front-running impurities, and then 14:1 methylene
chloride/isopropyl alcohol as an eluent to elute the
product to yield 650 mg (39%) of 6 as a cream-colored
solid: IR (CHCl3) 1772, 1743 cm-1; 1H NMR (400 MHz,
CDCl3) ? 1.12-1.22 (6H, m), 2.18-2.22 (2H, m), 2.52 (3H,
s), 2.60-2.68 (1H, m), 2.72-2.82 (1H, m), 2.93 (4H, s),
3.00-3.10 (2H, m), 3.38-3.58 (7H, m), 3.85-3.92 (1H,
m), 4.24-4.32 (2H, m), 5.63-5.71 (1H, m), 6.41 (1H, s),
6.86 (1H, s), 7.20-7.25 (3H, m), 7.36 and 7.57 (4H, AA¢
BB¢ q, J ) 8.8), 7.75 and 8.21 (4H, AA¢ BB¢ q, J ) 8.8);
MS, m/e 702 (M+). An almost equal amount of the dimer
was also obtained as a yellow oil: IR (CHCl3) 3445, 2780,
1653, 1627 cm-1; 1H NMR (CDCl3) ? 1.16 (3H, t, J ) 7.2),
Scheme 2. Synthesis of LSD Derivatives
898 Bioconjugate Chem., Vol. 8, No. 6, 1997 Li et al.
1.21 (3H, t, J ) 7.1), 2.15-2.23 (2H, m), 2.51 (3H, s),
2.56-2.64 (1H, m), 2.77 (1H, t, J ) 11), 2.96-3.03 (1H,
m), 3.03-3.09 (1H, m), 3.35-3.47 (5H, m), 3.47-3.55 (2H,
m), 3.80-3.86 (1H, m), 4.20-4.30 (2H, m), 5.90-5.96 (1H,
m), 6.85 (1H, s), 7.17-7.25 (3H, m), 7.45 and 7.54 (4H,
AA¢ BB¢q, J ) 8.1); MS, m/e 967 (M+H).
Scheme 3. Preparation of LSD Protein Conjugates
LSD Derivatives Bioconjugate Chem., Vol. 8, No. 6, 1997 899
Synthesis of 1-[[5-[8b-9,10-Didehydro-8-[(diethylamino)
carbonyl]ergolin-6-yl]-1,5-dioxopentyl]oxy]-2,5-pyrrolidinedione
(7, Scheme 3). A solution of 200 mg (0.65
mmol) of nor-LSD in 10 mL of anhydrous THF, under
argon, was treated with 161 mg (0.65 mmol) of 5-[(2,5-
dioxo-1-pyrrolidinyl)oxy]-5-oxopentanoyl chloride (20, 21),
followed by 0.2 mL (1.4 mmol) of anhydrous triethylamine.
The reaction mixture was stirred at room temperature
for 30 min and then concentrated at reduced
pressure. The residue was dissolved in methylene chloride,
washed with H2O and saturated aqueous sodium
bicarbonate solution, dried over anhydrous sodium sulfate,
and concentrated at reduced pressure to yield 330
mg (98%) of 7 as a yellow amorphous solid: UV (CH3-
OH) Ïmax 308 ( ) 8980); IR (KBr) 3396, 1814-1739, 1634,
1628 cm-1; 1H NMR (400 MHz, CDCl3) ? 1.08-1.18 (3H,
m), 1.25-1.38 (3H, m), 2.05-2.25 (2H, m), 2.43-3.25 (6H,
m), 2.83 (4H, s), 3.28-3.60 6H, m), [4.26 (d, J ) 13.6)
(major) and 5.02 (d, J ) 13.6) (minor)] (1H, rotamers),
[4.73-4.81 (minor) and 5.25-5.33 (major)] (1H, m, rotamers),
6.38 (1H, m), [6.90 (major) and 6.95 (minor)] (1H,
s, rotamers), 7.08-7.30 (3H, m), 8.00 (1H, m); MS, m/e
521 (M+H).
Synthesis of 8??-6-(3-Aminopropyl)-9,10-didehydro-N,Ndiethylergoline-
8-carboxamide (9, Scheme 2). Alkylation
of nor-LSD with iodopropylphthalimide was carried out
according to the procedure of Marzoni and Garbrect (16).
The resulting phthalimide derivative (820 mg, 1.65 mmol)
in 30 mL of methanol was treated with 0.4 mL (12.7
mmol) of anhydrous hydrazine and stirred at room
temperature overnight. The reaction mixture was filtered
and concentrated at reduced pressure. The residue
was chromatographed on 100 g of silica gel using 2%
triethylamine/15% methanol in methylene chloride as an
eluent. Fractions containing product were combined and
rechromatographed on 150 g of silica gel using 2%
triethylamine/15% methanol in chloroform as an eluent
to yield 560 mg (93%) of 9 as a yellow amorphous solid:
IR (CHCl3) 3479, 1663, 1624 cm-1; 1H NMR (400 MHz,
CDCl3) ? 1.17 (3H, t, J ) 7), 1.25 (3H, t, J ) 7), 1.70-
1.80 (1H, m), 1.83-1.93 (1H, m), 1.90 (2H, s), 2.60-2.73
(2H, m), 2.79-2.87 (1H, m), 2.90-2.99 (2H, m), 2.99-
3.09 (1H, m), 3.13-3.20 (1H, m), 3.25-3.33 (1H, m),
3.35-3.52 (6H, m), 6.28 (1H, s), 6.88 (1H, s), 7.10-7.17
(2H, m), 7.17-7.23 (1H, m), 8.20 (1H, br s); MS, m/e
366 (M+).
Synthesis of 1-[[[4¢-[[[3-[8??-9,10-Didehydro-8-[[(diethylamino)
carbonyl]ergolin-6-yl]propyl]amino]carbonyl]-1,1¢-
biphenyl]-4-yl]carbonyl]oxy]-2,5-pyrrolidinedione (10,
Scheme 2). A solution of 1.32 g (3.7 mmol) of 5 in 50 mL
of anhydrous methylene chloride under argon was cooled
to 0 °C and treated with a solution of 535 mg (1.46 mmol)
of 9 in 50 mL of anhydrous methylene chloride added
dropwise over a 30 min period. After the addition was
completed, the reaction mixture was washed with a
saturated aqueous sodium bicarbonate solution, dried
over anhydrous sodium sulfate, and concentrated under
vacuum. The residue was chromatographed on 100 g of
silica gel using 5% isopropyl alcohol as an eluent.
Fractions containing product were combined and concentrated
at reduced pressure to a yellow solid. The solid
was redissolved in ether and concentrated five times to
remove residual isopropyl alcohol to yield 280 mg (28%)
of 10 as a yellow solid: IR (CHCl3) 3479, 1773, 1743, 1636
cm-1; 1H NMR (400 MHz, CDCl3) ? 0.86 (3H, m), 1.11
(3H, t, J ) 6.9), 1.82-1.92 (1H, m), 1.93-2.10 (1H, m),
2.70-2.88 (4H, m), 2.93 (4H, s) 3.08-3.20 (2H, m), 3.22-
3.38 (4H, m), 3.40-3.60 (4H, m), 3.60-3.70 (1H, m),
3.98-4.08 (1H, m), 6.35 (1H, s), 6.93 (1H, s), 7.15-7.25
(3H, m), 7.61 and 7.95 (4H, AA¢ BB¢q, J ) 8), 7.67 and
8.19 (4H, AA¢ BB¢q, J ) 8.3), 8.00 (1H, s), 9.05 (1H, br
s); MS, m/e 688 (M+H); [R]D ) +9° (c 0.355%, CHCl3).
Preparations of Protein Conjugates. Synthesis of
1-[[[(4-Isothiocyanatophenyl)carbonyl]amino]butyl]-N,Ndiethyl-
d-lysergamide-BTG (4, Scheme 3). A solution of
698 mg of BTG in 20 mL of 50 mM KPi, pH 7.5, was
cooled to 0 °C and treated with 58 mL of DMSO, added
dropwise, very slowly over a 2 h period. The mixture was
treated with a solution of 90 mg (0.16 mmol) of 3 in 2
mL of DMSO, added dropwise very slowly. The reaction
mixture was stirred at room temperature for 18 h, poured
into a dialysis bag of 50 kDa molecular weight cutoff, and
dialyzed 108-fold in 50 mM KPi, pH 7.5. The resulting
conjugate was filtered through a 0.22 ?m sterile filter to
yield 116 mL of the LSD-BTG immunogen 4. The protein
concentration was determined to be 5.3 mg/mL. The
degree of drug substitution on the BTG protein was
determined by the ability of remaining uncoupled lysine
residues to react with TNBS (18, 19). Unmodified BTG,
at the same concentration as the conjugate, was treated
in the same manner with TNBS to provide a control. This
procedure produced a yellow complex with an absorbance
maximum at 325 nm and was used to calculate the drug
substitution expressed as percent modification. The
assay showed a 63.8% modification of available lysines
on BTG.
Synthesis of 5-[8??-9,10-Didehydro-8-[(diethylamino)-
carbonyl]ergolin-6-yl]-1,5-dioxopentyl-BTG (8, Scheme 3).
A solution of 700 mg of BTG in 13 mL of 50 mM KPi, pH
7.5, was cooled to 0 °C and treated with 13 mL of DMSO,
added dropwise, very slowly. After the addition was
complete, a solution of 84 mg (0.16 mmol) of 7 in 1 mL of
DMSO was added dropwise very slowly. The reaction
mixture was stirred at room temperature for 18 h, poured
into a dialysis bag of 50 kDa molecular weight cutoff, and
dialyzed 106-fold in 50 mM KPi, pH 7.5. The resulting
conjugate was filtered through a 0.22 ?m sterile filter to
yield the LSD-BTG conjugate 8. The protein concentration
was determined to be 12.1 mg/mL. The TNBS assay
showed a 45% modification of available lysines in BTG.
LSD Derivatives Exposed to Fluorescent Light,
Oxygen, and Different Solution pH Values. All LSD
derivatives (6, 7, and 10) were placed in quartz cuvettes,
and the irradiation experiments were conducted under
the following conditions: LSD derivatives were dissolved
in DMSO at a concentration of 1 mg/mL and further
diluted to 2.6 mM in 10 mM KPi buffer, pH 7.5, containing
0.09% NaN3, 5 mM EDTA, and 0.1% Tween 20. The
solutions were kept at 2-8 °C in the dark for 3 days to
ensure a complete hydrolysis of the NHS ester (e.g. 10a,
Scheme 2). This is indicated by a complete disappearance
of the starting material by TLC examination (data
not shown). For the stability to light study, samples were
exposed to a 20 W desk fluorescent light at room
temperature. The distance between the fluorescent light
source and the experimental samples was 15 cm. The
experimental samples were subjected to irradiation for
various times, and then fluorescence from the samples
was measured, with Ïex ) 320 nm and Ïem ) 445 nm.
Triplicate measurements were performed under each
condition. Coefficients of variation were found to be
e1.5% for the analytical method. From these data we
established that >9% loss in fluorescent intensity represented
significant decomposition of the tested compounds.
This change of 9% represents three standard
deviations from the mean, which is a >99% confidence
interval. Corresponding LSD derivatives kept in the
dark, at room temperature, were used as controls. These
experiments were designed to investigate the effect of
indoor light on the stability of the C-9,10 double-bond
900 Bioconjugate Chem., Vol. 8, No. 6, 1997 Li et al.
position of LSD derivatives and to rank the light stabilities
of these derivatives.
For stability to oxygen and solution pH studies,
samples were placed in amber glass bottles and saturated
with either oxygen or argon. Subsequently, these experimental
samples were incubated at 45 °C for 10 days.
Samples kept at 2-8 °C in the dark with argon were
classified as the control. These experiments were conducted
to explore the effect of oxygen, pH, and heat on
the stability of the C-9,10 double-bond position of LSD
derivatives and to rank the stability of these derivatives
under these conditions.
Antibody Generation. Several goats were placed on
a modified immunization program, as described by
Vaitukaitis (22) using LSD immunogens (4 or 8). Briefly,
immunogen 4 or 8 was mixed with Freund??s adjuvant,
and 1 mg of the immunogen containing complete Freund??s
was injected into multiple sites across the back of
each goat. At week two, each goat continued to receive
1 mg of the immunogen containing incomplete Freund??s.
This injection was repeated twice at 1 week intervals,
followed by a monthly injection of 0.5 mg of the immunogen
mixed with incomplete Freund??s adjuvant for a
period of 6 months.
The individual animals were monitored for antibody
titer and for cross-reactivity with LSD, nor-LSD, and
2-oxo-3-hydroxy-LSD by an ELISA method. Specifically,
a selected derivative was covalently coupled to a carrier
protein (ovalbumin). Polystyrene 96 well microtiter
plates were coated with 50 ?L of a 1.6 ?g/mL LSD-ovalbumin
conjugate in PBS buffer (50 mM KPi, pH 7.2, containing
150 mM NaCl) and allowed to incubate for 2 h
at room temperature or overnight at 2-8 °C. The plates
were washed with PBS buffer and blocked with 1% BSA
in PBS buffer. Fifty microliters of LSD, nor-LSD, or
2-oxo-3-hydroxy-LSD diluted in 1% BSA/PBS buffer at
various concentrations or 50 ?L of 1% BSA/PBS buffer
without the drug as a control was added into each well.
Fifty microliters of the appropriate antiserum in 1% BSA/
PBS buffer was then added to each well. The plates were
incubated for 1 h at 37 °C and then washed with PBS/
Tween 20 buffer. Anti-goat-alkaline phosphatase conjugate
and p-nitrophenyl phosphate were then used to
generate a detection signal. Two criteria were used to
select antibodies: (1) affinity of antibodies as estimated
by IC50; (2) inhibition of solid-phase antibody binding by
soluble LSD and its major metabolites, namely, nor-LSD
and 2-oxo-3-hydroxy-LSD. Once several animals were
selected from an immunogen, a pool of antiserum was
made and used to develop the immunoassay.
Preparation of LSD-Ovalbumin Conjugate (11,
Scheme 3). Seventeen and a half milliliters of ovalbumin
solution at 25 mg/mL in 50 mM KPi buffer, pH 7.5,
was cooled in an ice bath, and to this was slowly added
10 mL of DMSO. Seven and a half miliigrams of selected
LSD derivative was then dissolved in 1.5 mL of anhydrous
DMSO to make a 5 mg/mL solution. The LSD
derivative solution was added dropwise into the ovalbumin
solution with stirring, and stirring was continued
for 18 h at room temperature. The resulting LSDovalbumin
conjugate was dialyzed 1012-fold using 30 kDa
molecular weight cutoff dialysis bags. The final total
protein concentration of LSD-ovalbumin conjugate was
determined according to the Bradford protein assay (15).
Characterization of LSD-Ovalbumin Conjugate.
To determine the concentration of the noncovalently
bound LSD in the LSD-ovalbumin conjugate, the following
experiments were conducted: 1.5 mL of 12.5 mg/
mL LSD-ovalbumin conjugate in KPi buffer, pH 7.5, was
heat stressed at 45 °C in a white nontransparent polyethylene
container (HDPE) for 24 h. It was then immediately
mixed with 1.5 mL of 40% DMF in 10 mM KPi,
pH 7.5. The material was then left at room temperature
for 2 h, placed in a ovalbumin precoated Centricon filter
(Amicon, Beverly, MA) with a molecular weight cutoff of
30 kDa, and centrifuged at 600g for 2 h. The resulting
filtrate was then analyzed by fluorescence spectroscopy.
Completely hydrolyzed LSD-biphenyl-NHS (6) solutions
with concentrations ranging from 200 to 3200 ng/mL
were used as fluorescence standard, and a linear regression
method was used to generate a calibration curve.
The concentration of LSD in the filtrate was derived from
the standard curve. Filtrate from ovalbumin treated according
to the same method as the LSD-ovalbumin conjugate
was used as the control for background measurements.
A method was also designed to measure the total
number of LSD derivatives per ovalbumin in the LSDovalbumin
conjugate. The LSD-ovalbumin conjugate
was diluted to a concentration of 0.125 mg/mL of total
protein, and the fluorescence at 445 nm from the diluted
conjugate solution was measured. Ovalbumin treated
according to the same method as the LSD-ovalbumin
conjugate was used as the control for a blank measurement.
Completely hydrolyzed LSD-biphenyl-NHS (6)
solution concentrations ranging from 200 to 3200 ng/mL
were again used as fluorescence standards to generate a
calibration curve. Total concentration of LSD molecules
per ovalbumin was estimated from the calibration curve.
Preparation of the LSD Microparticle (12, Scheme
3). Ten milliliters of carboxyl-modified microparticle
(10% solids) was first washed by centrifugation at 10000g
with 0.1% Tween 20 in H2O. To each milliliter of
particles was added 20 mL of 0.1% Tween 20 in water,
the mixture was centrifuged and decanted, and the
particles were subsequently resuspended. This process
was repeated five times, and the microparticle concentration
was then adjusted to 3% (w/v) with a 0.1% Tween
20 solution. One and two-tenths milliliters of NHB (25
mg/mL, 0.37 mmol), previously dissolved in DMSO, was
then added slowly to the 30 mL of microparticle suspension,
under rapid stirring conditions, and the suspension
was stirred for 10 min at 25 °C. To this suspension was
added 1.7 mL of a freshly prepared CMC solution (50 mg/
mL, 0.34 mmol), and the mixture was stirred slowly for
3 h at 25 °C. The material was then washed according
to the method of centrifugation described above. The
washed, activated microparticles (45 mL at 2%) were
immediately mixed with LSD-ovalbumin/ovalbumin mixture
at different molar ratios (total protein concentration
was fixed at 3.1 mg/mL) diluted in 50 mM sodium
bicarbonate buffer, pH 8.6, and this mixture was allowed
to stir for 15 h at 25 °C. The resulting LSD microparticles
were then washed according to the method of
centrifugation described above using a wash solution of
10 mM KPi buffer, pH 7.5, containing 0.09% NaN3 and
0.1% Tween 20. The washed microparticle was then
resuspended in this buffer at 1.0% solids (w/v).
Development of the LSD Immunoassay. The LSD
immunoassay contains three reagents: (1) the antibody
reagent, which was made by placing the titered antibody
in a solution of 50 mM HEPES, pH 6.5, containing 0.1%
BSA protein, 0.5% NaCl, and 0.09% NaN3; (2) a reaction
buffer containing 50 mM PIPES, pH 7.0, with 2-3%
PEG, 2% NaCl, and 0.09% NaN3; and (3) a LSD microparticle
reagent, diluted from 1% stock solution to 0.2%
solids in a buffer containing 10 mM KPi, pH 7.5, 0.09%
NaN3, and 0.1% Tween 20. In addition, LSD calibrators
at concentrations between 0 and 1 ng/mL in normal urine
containing 0.09% NaN3 were used. The concentration of
LSD Derivatives Bioconjugate Chem., Vol. 8, No. 6, 1997 901
these LSD standards were verified by GC/MS/MS method.
The antibody concentration was adjusted so that the
agglutination of the LSD microparticles was inhibited
proportionally to the LSD concentration in the calibrators.
The light scattering difference between different
calibrators was also maximized in the calibration range
(0-1 ng/mL) to obtain maximum sensitivity.
Cross-reactivity to structurally related compounds was
conducted as follows: Normal human urine samples were
spiked with the structurally related compound of LSD
at various concentrations and tested as unknowns in the
OnLine assay. The percent cross-reactivity of a structurally
related compound was determined using the concentration
of the compound that provided displacement
equivalent to 0.5 ng/mL (1.5 nM) LSD.
One thousand presumed negative urine specimens
were obtained from a large drug abuse screening laboratory.
These samples had been previously screened and
found to be negative for the SAMHSA five panel (cannabinolds,
opiates, cocaine metabolite, amphetamine, and
phencyclidine). At the time of analysis, these samples
were simultaneously screened for LSD with the Abuscreen
RIA and OnLine assay. The Abuscreen RIA has a
>99.5% accuracy rate and was used as a reference
method in addition to GC/MS/MS method. In addition,
LSD positive samples were supplied by Dr. R. Foltz and
Dr. D. Kuntz (Northwest Toxicology, Salt Lake City, UT).
These samples had been previously screened positive by
the Abuscreen RIA and were subsequently confirmed by
GC/MS/MS. These samples were received frozen and
were stored at -20 °C until the day of analysis. For analysis,
a qualitative screening assay was performed as
follows: a single point calibration standard was used, and
its absorbance value was assigned as the cutoff value. A
positive result was reported if the sample absorbance
value was greater than or equal to the absorbance of the
cutoff calibrator.
RESULTS AND DISCUSSION
Synthesis and Stability Studies of the LSD Derivatives.
A detailed analytical study of the synthesized
LSD derivatives was necessary to determine which
derivatives should be used to develop the LSD OnLine
assay. The effect of exposure to fluorescent light on the
LSD derivatives is shown in Figure 1. The C-9,10 double
bond of LSD as previously reported can undergo photocatalytic
hydration (5). A potential structure change or
instability at this position was indicated by a change in
fluorescence intensity when compared to a control. It
was demonstrated that the fluorescence intensity of each
compound decreased as the exposure time to fluorescent
light increased. These results suggested that the order
of stability of the LSD derivatives to photon-catalyzed
hydration is LSD-biphenyl-COOH (hydrolyzed from 6) >
nor-LSD-biphenyl-COOH (hydrolyzed from 10) > nor-
LSD-aliphatic-COOH (hydrolyzed from 7). It is hypothesized
that the improved stability of 6 may come from
the presence of biphenyl group at the N-1 position of the
LSD molecule, where the biphenyl moiety can effectively
stack on the LSD and exclude water molecules from
interaction, thus stabilizing the photolabile C-9,10 center.
Such hydrophobic stacking interaction has been documented
(23, 24). When the biphenyl modification was
at position 6 of the LSD molecule, the biphenyl linker
could not effectively stack on the LSD and the protection
efficiency was reduced. The aliphatic moiety at position
6 would be predicted to offer little or no protection effect
to the LSD molecule, and indeed, this molecule had the
least stability under these conditions. The LSD-biphenyl
compound (6) was chosen for the construction of the
conjugate used in the nonisotopic immunoassay. This
compound displayed a 50-60% decomposition after having
been exposed to the described conditions for 4 days.
To ensure good assay stability, this issue was addressed
by placing the microparticle reagent containing this
derivative in a nontransparent polyethylene container.
An accelerated stability study indicated that this container
protects the reagent from light to ensure <10%
loss at normal room light conditions for 1 year. Further
data to emphasize the need not to have decomposition
are reflected in cross-reactivity studies of antibodies
generated from LSD immunogen (4) to completely photodecomposed
LSD-biphenyl-COOH (6). It was found
that the cross-reactivity of this decomposed material was
<20% (data not shown), which would cause serious loss
in assay sensitivity if the derivative was allowed to
decomposed.
Under heating conditions, oxygen may have an effect
on the C-9,10 double bond (25). However, it appears from
our experiments that oxygen did not affect the stability
of the C-9,10 double bond for the LSD derivatives. Since
protons can catalyze the hydration of the C-9,10 double
bond (5, 25), we explored the effect of pH on the hydration
of different LSD derivatives at high temperatures. Solution
pH was chosen to be 6.0 or 7.5, because this is the
most acceptable pH range for immunochemical reactions.
At pH 6.0, in the presence or absence of oxygen, after 10
days at 45 °C, compound 10 lost 20% of its fluorescence
intensity, while fluorescence intensity changes for compounds
6 and 7 were negligible. No significant changes
in fluorescence intensity were observed for all three
compounds when they were kept at pH 7.5 for 10 days
at 45 °C. The results suggested that the LSD derivatives
were more stable in pH 7.5 buffer than in pH 6.0 buffer.
Synthesis of LSD Immunogen and the Generation
of LSD Antibodies. Since only 1% of ingested LSD
is excreted in urine and the typical ingested dose is very
low, it is prudent to generate LSD antibodies that can
recognize not only LSD but also LSD metabolites, yet
avoid other undesirable ergot alkaloids compounds.
Figure 1. Effect of fluorescent light irradiation on the stability
of LSD derivatives. Structural changes of LSD derivatives at
the C-9,10 position were indicated by changes in fluorescence
intensity. Condition: 2.6 mM LSD derivatives in 10 mM KPi,
pH 7.5, buffer containing 5 mM EDTA, 0.09% NaN3, and 0.1%
Tween 20. All experiments were conducted at 25 °C. Corresponding
LSD derivatives kept in the dark at 25 °C were used
as controls. The Y axis represents the fluorescence intensity of
irradiated LSD derivatives when compared to their controls. The
X axis is exposure time to fluorescent light. Error bar represents
( 2 SD.
902 Bioconjugate Chem., Vol. 8, No. 6, 1997 Li et al.
Selectivity of an antibody to a hapten can be directed
such that the antibody preferentially recognizes the part
of the molecule that is farthest away from the attachment
of the hapten to a carrier protein (26). It was assumed
that this will allow the antibody to tolerate changes to
the hapten near its point of attachment to the carrier
protein. Under this hypothesis, we designed two immunogens
(4 and 8). These immunogens directed antibodies
to be less specific regarding changes near the indole ring
of LSD (27) or at position 6 of LSD; therefore, crossreactivity
toward 2-oxo-3-hydroxy-LSD, 13-hydroxy-LSD,
14-hydroxy-LSD, and nor-LSD would be predicted to be
higher for antibodies generated with immunogen 4.
Antibodies generated from immunogen 8 would be expected
to have high cross-reactivity toward 2-oxo-3-
hydroxy-LSD and nor-LSD.
LSD-biphenyl-NHS (6) was used to construct the LSDovalbumin
conjugate label for the ELISA method. The
binding between the LSD-ovalbumin conjugate label and
the antibodies generated from immunogen 4 can be
displaced with LSD (100%), nor-LSD (20%), and 2-oxo-
3-hydroxy-LSD (50%). When antibodies generated from
immunogen 8 were used, the binding between the LSDovalbumin
conjugate label and antibodies could be displaced
by LSD and had high cross-reactivity to nor-LSD
(40%) but had <20% cross-reactivity to 2-oxo-3-hydroxy-
LSD. From the cross-reactivity studies, antibodies raised
against immunogen 4 were selected, and, from the
derivative stability studies, derivative 6 was chosen for
development of a LSD immunoassay.
Preparation and Characterization of LSD-Ovalbumin
Conjugate. For a LSD assay, a 1:1 molar ratio
between LSD-biphenyl-NHS (6) and ovalbumin was used
to synthesize the LSD-ovalbumin conjugate stock solution.
Since the LSD derivative is a highly hydrophobic
compound, it can be trapped in ovalbumin or absorbed
on the ovalbumin surface noncovalently. This unbound
LSD derivative could be gradually released from the
conjugate and could cause poor stability. The unbound
LSD derivative could react with titered antibody and
reduce the sensitivity of the LSD assay. Therefore, it
was important to develop a reproducible dialysis procedure
to remove the unbound LSD from the LSD-ovalbumin
conjugate. It was also important to establish a
method to measure the noncovalently bound LSD derivatives
on the conjugate so that methods could be selected
that prevent this from occurring. To accomplish this, the
conjugate was denatured at 45 °C and then in 20% DMF
solvent to release any noncovalently bound LSD derivative.
Under these mild denaturing conditions, no protein
precipitation was observed. Our results indicated that
free LSD derivative accounted for <0.3% of total LSD
derivative loaded on ovalbumin when an extensive
dialysis (1012-fold) procedure was used. This procedure
was necessary to obtain a stable OnLine LSD assay
reagent.
Methods were also developed to quantify the number
of the LSD molecules per ovalbumin molecule in the
Figure 2. Representative concentration response curve on the
Olympus AU800 analyzer of the Abuscreen LSD OnLine assay.
Absorbance (ABS) is expressed as the milliabsorbance multiplied
by a factor of 10. Error bar represents ( 1 SD.
Table 1. Qualitative Precision of Abuscreen LSD OnLine
Assaya
a The Olympus AU 800 analyzer multiplies the milliabsorbance
units by a factor of 10 to report results. Precision was determined
to be <3.0% at 0 ng/mL (blank) urine.
Table 2. Cross-Reactivities of Abuscreen OnLine LSD
Assay
LSD Derivatives Bioconjugate Chem., Vol. 8, No. 6, 1997 903
LSD-ovalbumin conjugate stock solution. Since amino
groups at the protein surface are used in the coupling,
normally, the degree of drug substitution can be determined
by the ability of remaining uncoupled amine
residues that react with TNBS. However, since the LSD
substitution ratio was low (mean < one LSD per ovalbumin),
poor results were obtained using the TNBS
method. It was found that the number of LSD molecules
in the conjugate could be estimated directly using the
LSD fluorescence intensity. The fluorescence from ovalbumin
at the same concentration as the conjugate was
insignificantly small compared to the total intensity.
Using completely hydrolyzed LSD-biphenyl-NHS (6) as
a standard, the conjugates have been shown to contain
0.6-0.8 LSD molecules per ovalbumin molecule. Because
coupling of LSD to ovalbumin would change the fluorescence
quantum yield from LSD, this method only provided
an estimate of the loading of LSD molecules per
ovalbumin and was useful for quality control purposes
(data not shown).
Synthesis and Characterization of LSD-Microparticle
Conjugate. Several different molar ratios of
the drug protein conjugate to microparticle were evaluated
to determine the optimal ratio that produced the
best dose response curve. In the development of a
microparticle-based immunoassay, it is important that
proper agglutination occurs in the absence of free antigen.
To accomplish this, proper amounts of drug protein
conjugate must be coupled to each microparticle such that
an equivalence point can be reached, allowing the crosslinking
of microparticles by antibody. Excess antigen or
excess antibody in the system will prevent the formation
of the large aggregates produced by cross-linking.
To establish the proper substitution of drug onto
microparticle, LSD was first covalently coupled to the
ovalbumin protein (stock conjugate) followed by mixing
of the LSD-ovalbumin conjugate with ovalbumin at
various molar ratios and coupling the LSD-ovalbumin/
ovalbumin mixture to the microparticle. Each coupled
microparticle was then titrated against the antibody to
determine the performance of each LSD-ovalbumin/
ovalbumin molar ratio. The ratio that gave the greatest
dose response curve and the lowest nonspecific binding
(agglutination rate in the absence of antibody) was
selected. This was determined to be a molar ratio of 1:8
(LSD-ovalbumin/ovalbumin).
Development of LSD Assay. Using an endpoint
analysis reading at 520 nm, a dose response curve was
generated with various concentrations of LSD as shown
in Figure 2. The light scattering difference measured by
light transmission from 0 ng/mL to the cutoff concentration
of LSD (0.5 ng/mL) was >130 milliabsorbances (mA);
the overall difference from 0 to 1.0 ng/mL was >240 mA.
Table 1 shows that the qualitative intra-assay (n ) 20)
and interassay (n ) 100) precision had CVs of <5%.
Table 2 illustrates the cross-reactivity of the LSD
OnLine assay to structurally related compounds of LSD.
As expected, this assay had low cross-reactivity to iso-
LSD (2.4%, molar concentration); the cross-reactivity to
nor-LSD was 25% and to 2-oxo-3-hydroxy-LSD was 32%.
The cross-reactivities of other structurally related compounds
that are undesirable to detect, such as serotonin,
tryptophan, ergotamine, egonovine, and others, were
<0.002%. Finally, the limit of detection (LOD) of the
assay was determined by performing 20 replicate assays
on the 0 ng/mL calibrator. Two standard deviations
below the mean yields a LOD of <0.2 ng/mL LSD.
Table 3 shows the correlation of the OnLine LSD
screening assay with RIA and GC/MS/MS methods. GC/
MS/MS confirmed LSD positive clinical samples were
used to study patient correlation. Eighty-one positive
samples were tested in the OnLine LSD assay. The
distribution of LSD concentration in these samples is also
shown in Table 3. Twenty percent of the samples
Table 3.
904 Bioconjugate Chem., Vol. 8, No. 6, 1997 Li et al.
contained <0.5 ng/mL of LSD according to GC/MS/MS
data. Due to the high cross-reactivity to major LSD
metabolites, all of these samples were positive by OnLine
LSD assay. One thousand presumptive negative samples
were also tested; 993 were negative and 7 were positive.
All of the presumptive negative samples were found to
be negative by RIA. These seven samples that were
OnLine positive and RIA negative were found to be GC/
MS/MS negative.
CONCLUSION
A homogeneous microparticle-based immunoassay has
been developed for the detection of LSD in human urine
with the required sensitivity and specificity. Three major
issues were considered when this assay was developed:
(1) the stability of the LSD derivatives; (2) the stability
of LSD microparticles; (3) the cross-reactivity of antibodies.
Light, temperature, and solution pH can alter the
structure of LSD at the C-9,10 double-bond position.
Therefore, it is desirable to prepare and select a derivative
that generates a stable LSD microparticle which is
able to withstand long-term storage. On the basis of
stability studies, we have selected LSD-biphenyl-NHS (6)
as the best derivative for the development of the LSD
OnLine assay. Besides a stable LSD derivative, LSD
microparticles free of noncovalently bound LSD are
necessary for the OnLine technology to obtain the
required assay sensitivity and reagent stability. The
conjugation and dialysis procedures reported here have
allowed us to minimize unbound LSD in the LSD microparticle
reagent and to achieve the targeted sensitivity
and stability of the immunoassay. Due to the extent of
LSD in vivo metabolism and low ingestion dosage, the
concentration of parent compound (LSD) in urine is
extremely low. To overcome this, we designed and
selected an immunogen using a LSD analogue derivatized
through the indole nitrogen and conjugated the
derivative to BTG. The antibody generated by this
immunogen has demonstrated broad reactivity toward
LSD and several LSD metabolites. All of these factors
were essential in the successful development of the LSD
OnLine assay, which demonstrated excellent clinical
sensitivity.
ACKNOWLEDGMENT
We thank Dr. D. Kuntz and Dr. R. Foltz for providing
the GC/MS/MS data for all of the LSD-positive samples
used in this study and Mr. E. Nowaswiat for providing
compounds 1a and 1b. We also thank Dr. L. Arabshahi,
Ms. L. Allison, Dr. K. Savoca, and Dr. K. Schwenzer for
their technical help during the development of this assay.
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