Pharmacology of LSD
Dolder et al. supplementary material
SUPPLEMENTARY METHODS
Analysis of LSD and O-H-LSD
LSD and O-H-LSD concentrations were determined in plasma and urine on an ion trap LCMS/MS instrument (LTQ XL mass spectrometer from Thermo Scientific, Basel, Switzerland)
coupled to a turbulent-flow extraction system. The method has been reported in detail
elsewhere (Dolder et al., 2014) and was validated according to FDA criteria for the
determination of serum and urine concentrations of LSD and O-H-LSD. Because plasma
samples were analyzed in the present study, a partial validation based on the method for
serum (Dolder et al., 2014) was performed for the analysis of LSD and O-H-LSD in plasma.
The method is summarized below.
Standards: LSD and LSD-d3 were obtained from Lipomed (Arlesheim, Switzerland) and 2oxo-3-hydroxy LSD (O-H-LSD) from Cerilliant (Round Rock, TX, US). Equipment: The
sample extraction system (Transcend TLX1 HPLC, Thermo Scientific, Basel Switzerland)
consisted of a Thermo PAL autosampler and two Accela 600 pumps as eluting and loading
pumps. A cyclone and a C18XL turboflow column (Thermo Scientific, Basel Switzerland) for
extraction, and a 3 µm Betasyl/Phenyl/Hexyl column (Thermo Scientific, Basel, Switzerland)
for chromatographic separation were used. The online extraction system was coupled to a
LTQ XL mass spectrometer from Thermo Scientific (Basel, Switzerland) using atmospheric
pressure ionization (APCI). LC method: Four mobile phases were used in gradient mode
based on a previously published method (Mueller et al., 2011). For extraction, loading B
consisted of 10 mM ammonium carbonate in water, eluting A was 5 mM ammonium acetate
in water containing 0.1% formic acid and eluting B 5 mM ammonium acetate in methanol
containing 0.5% formic acid, respectively. Loading B was used as alkaline loading buffer,
eluting A and B for chromatographic separation. Loading and eluting C (acetonitrile
/acetone/2-propanol, 1:1:1 (V/V/V) were used to clean the extracting and the analytical
columns. MS conditions: For the quantification of LSD and its metabolite, APCI was used as
the ionization source in positive ion mode. LSD and O-H-LSD were quantified using single
reaction monitoring (SRM) of the corresponding mass transitions (LSD m/z 324.6  223.23,
O-H-LSD m/z 356.33  338.33, LSD-d3 327.21 226.2). Sample preparation:
Acetonitrile for protein precipitation (100 µl) and 10 µl of a LSD-d3 internal standard
solution (100 ng/ml) were added to 100 µl of plasma. 100 µl of urine were diluted with 50 µl
of an ammonium acetate buffer (50mM, pH4) and 10 µl of LSD-d3 internal standard solution
1
Pharmacology of LSD
Dolder et al. supplementary material
was added. No significant matrix effects and no ion suppression were observed for serum or
urine. Selectivity, matrix effects, and recovery. Confirming selectivity, none of five blank
serum, plasma or urine samples from different patients showed any interference within the
measured mass range and time frame. Recoveries in serum were 64.0% for LSD and 32.0%
for O-H-LSD. Recoveries were not determined for plasma. Recoveries in urine were 90% for
LSD and 88% for O-H-LSD.
Calibration in plasma: The calibration curves for LSD and O-H-LSD in plasma were linear
over the concentration range of 0.1 ng/ml to 10 ng/ml (both R2>0.99). Accordingly the lower
level of quantification was 0.1ng/ml for both LSD and O-H-LSD in plasma as identically
reported for serum (Dolder et al., 2014). Six quality control samples across the calibration
range (0.1-10 ng/ml for both LSD and O-H-LSD) were used with every run to demonstrate
accuracy and precision of the method for plasma. Mean intraday accuracy and precision for
LSD were 104% and 5. 5%, respectively, in plasma. Mean interday accuracy and precision for
LSD were 105% and 3.8%, respectively, in plasma. Mean intraday accuracy and precision for
O-H-LSD were 94.4% and 7.2%, respectively, in plasma. Mean interday accuracy and
precision for O-H-LSD were 96.2% and 7. 5%, respectively, in plasma. Intraday and interday
accuracy and precision values for each concentration of LSD and O-H-LSD are shown in
Table S1 and Table S2, respectively.
Calibration in urine: The calibration curves for LSD and O-H-LSD in urine were linear over
concentration ranges of 0.1 ng/ml to 33 ng/ml and 1.5 ng/ml to 333 ng/ml (both R2>0.99),
respectively. The lower levels of quantification were 0.1ng/ml and 1.5 ng/ml for LSD and OH-LSD, respectively, in urine (Dolder et al., 2014). Quality control samples (LSD: 0.1, 0.25,
0.6, 3.3, and 33 ng/ml; O-H-LSD: 1.5, 2.5, 6, 33, and 333 ng/ml) in urine were used with
every run to demonstrate accuracy and precision of the method. Mean intraday accuracy and
precision for LSD were 101% and 6.8%, respectively, in urine. Mean interday accuracy and
precision for LSD were 100% and 8.3%, respectively, in urine. Mean intraday accuracy and
precision for O-H-LSD were 97.5% and 5.0%, respectively, in urine. Mean intraday accuracy
and precision for O-H-LSD were 101% and 8.1%, respectively, in urine (Dolder et al., 2014).
REFERENCES
2
Pharmacology of LSD
Dolder et al. supplementary material
Dolder PC, Liechti ME, Rentsch KM (2014) Development and validation of a rapid turboflow
LC-MS/MS method for the quantification of LSD and 2-oxo-3-hydroxy LSD in serum
and urine samples of emergency toxicological cases. Anal Bioanal Chem
doi:10.1007/s00216-014-8388-1.
Mueller DM, Duretz B, Espourteille FA, Rentsch KM (2011) Development of a fully
automated toxicological LC-MS(n) screening system in urine using online extraction
with turbulent flow chromatography. Anal Bioanal Chem 400:89-100.
3
Pharmacology of LSD
Dolder et al. supplementary material
Supplementary Table S1. Intraday Precision and Accuracy Data of LSD and 2-oxo-3-hydroxy
LSD Measured in Plasma at Different Concentrations
LSD
Weighed-In
Concentrati
on (ng/mL)
Plasma
Measured
Concentration
(ng/mL)
Plasma
0.1
0.4
0.8
4
8
10
0.1±0.0
0.4±0.0
0.8±0.0
4.1±0.2
8.6±0.3
10.5±1.0
Mean Precision
SD (%)
Mean Accuracy
± SD (%)
Plasma
(n=6)
5.6
4.6
3.5
5.4
3.9
9.8
Plasma
(n=6)
105±5.9
102±4.7
101±3.5
103±5.6
107±4.1
105±10
O-H-LSD
0.1
0.1±0.0
10.4
94.4±9.4
0.4
0.4±0.0
6.4
102±6.5
0.8
0.8±0.1
9.2
96.9±8.9
4
3.6±0.2
5.7
91.1±5.2
8
7.5±0.6
7.6
94.3±7.1
10
8.8±0.4
4.0
88.0±3.5
Abbreviations: LSD, lysergic acid diethylamide; O-H-LSD, 2-oxo-3-hydroxy-LSD.
Supplementary Table S2. Interday Precision and Accuracy Data of LSD and 2-oxo-3-hydroxy
LSD Measured in Plasma at Different Concentrations
LSD
Weighed-In
Concentratio
n (ng/mL)
Plasma
Measured
Concentration
(ng/mL)
Plasma
0.1
0.4
0.8
4
8
10
0.1±0.0
0.4±0.0
0.8±0.0
4.1±0.2
8.4±0.3
10.3±0.3
Mean Precision
SD (%)
Mean Accuracy
± SD (%)
Plasma
(n=6)
Plasma
(n=6)
3.6
6.5
2.5
4.0
3.1
3.3
107±3.8
104±6.7
108±2.7
103±4.1
105±3.3
103±3.4
O-H-LSD
0.1
0.1±0.0
10.5
91.2±9.6
0.4
0.41±0.0
9.4
102±6.5
0.8
0.8±0.0
3.4
102±9.6
4
3.8±0.3
6.5
94.9±6.2
8
7.8±0.7
9.1
97.6±8.8
10
9.9±0.6
5.8
98.6±5.7
Abbreviations: LSD, lysergic acid diethylamide; O-H-LSD, 2-oxo-3-hydroxy-LSD.
4
Pharmacology of LSD
Dolder et al. supplementary material
Supplementary Results
S 1 , m a le
* *
*
L S D ( n g /m l)
*
*
+
*
1
t1 /2
-1 2
t 1 /2
te r m
S 2 , m a le
= 4 .8
10
= 7 .1
**
+
+
0 .1
L S D ( n g /m l)
10
0 1 2 3 4
6
8
10
12
16
= 2 .9
= 1 0 .6
*
*
1
*
+
*
+
12
16
+
0 .1
0 1 2 3 4
24
6
8
10
S 4 , m a le
S 3 , fe m a le
t1 /2
-1 2
t 1 /2
te r m
= 3 .5
10
= 7 .5
*
*
*
*
1
+
*
+
+
0 .1
L S D ( n g /m l)
* *
24
tim e (h )
10
L S D ( n g /m l)
te r m
*
tim e (h )
0 .0 1
*
*
*
1
t1 /2
-1 2
t 1 /2
te r m
= 3 .6
= 2 2 .1
+
*
+
+
12
16
24
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
16
24
0 1 2 3 4
6
8
10
tim e (h )
tim e (h )
S 6 , m a le
S 5 , fe m a le
t1 /2
10
t 1 /2
*
= 4 .3
-1 2
te r m
10
* **
*
= 1 9 .7
*
*
*
1
*
+
*
+
+
0 .1
L S D ( n g /m l)
* **
L S D ( n g /m l)
-1 2
t 1 /2
0 .0 1
0 .0 1
0 .0 1
*
*
1
t1 /2
-1 2
t 1 /2
te r m
= 4 .9
= 1 0 .9
+
*
+
+
16
24
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
16
24
0 1 2 3 4
6
8
10
tim e (h )
12
tim e (h )
S 7 , m a le
10
*
*
*
1
+
*
t1 /2
-1 2
t 1 /2
te r m
S 8 , m a le
= 3 .6
10
= 1 5 .7
* *
+
+
0 .1
0 .0 1
L S D ( n g /m l)
*
L S D ( n g /m l)
t1 /2
*
+
*
1
+
*
t1 /2
-1 2
t 1 /2
te r m
= 3 .9
= 1 1 .3
+
*
+
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
tim e (h )
16
24
0 1 2 3 4
6
8
10
12
16
24
tim e (h )
5
Pharmacology of LSD
Dolder et al. supplementary material
S 9 , fe m a le
10
L S D ( n g /m l)
*
+
*
+
*
1
-1 2
t 1 /2
te r m
S 1 0 , f e m a le
= 4 .3
10
= 4 .2
*
+
*
+
+
0 .1
L S D ( n g /m l)
+
t1 /2
0 1 2 3 4
6
8
10
12
16
= 3 .5
= 7 .7
*
*
1
+
*
+
+
0 .1
0 1 2 3 4
24
6
8
10
12
16
S 1 1 , f e m a le
*
*
*
+
*
1
+
*
t1 /2
-1 2
t 1 /2
te r m
S 1 2 , m a le
= 3 .6
10
= 5 .1
+
+
0 .1
L S D ( n g /m l)
**
24
tim e (h )
10
L S D ( n g /m l)
te r m
*
tim e (h )
+
+
*
*
1
+
*
+
*
+
*
0 .1
t1 /2
-1 2
t 1 /2
te r m
= 2 .2
= 2 .2
+
*
0 .0 1
0 .0 1
0 1 2 3 4
6
8
10
12
16
0 1 2 3 4
24
6
8
10
12
16
S 1 3 , m a le
10
+
*
1
t1 /2
-1 2
t 1 /2
te r m
= 2 .1
S 1 4 , f e m a le
t 1 / 2 - 1 2 = 4 .6
10
t 1 /2
= 2 .1
te r m
= 5 .6
+
*
+
*
L S D ( n g /m l)
+
*
24
tim e (h )
tim e (h )
L S D ( n g /m l)
-1 2
t 1 /2
0 .0 1
0 .0 1
+
*
+
*
0 .1
1
+
*
+
*
+
12
16
0 .1
0 .0 1
0 .0 1
0 1 2 3 4
6
8
10
12
16
0 1 2 3 4
24
6
8
10
S 1 6 , f e m a le
S 1 5 , f e m a le
t 1 / 2 - 1 2 = 2 .8
10
t 1 /2
*
*
+
*
1
te r m
10
= 4 .1
t1 /2
-1 2
t 1 /2
te r m
= 3 .1
= 6 .7
* *
+
*
*
+
*
L S D ( n g /m l)
**
24
tim e (h )
tim e (h )
L S D ( n g /m l)
t1 /2
+
*
+
0 .1
0 .0 1
*
1
*
+
*
+
+
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
tim e (h )
16
24
0 1 2 3 4
6
8
10
12
16
24
tim e (h )
Supplementary Figure S1. Semilogarithmic plots of the individual lysergic acid
diethylamide (LSD) plasma concentration-time profiles of all 16 individual curves. Each
panel represents data from 1 subject. Filled circles were used for male subjects and open
circles for female subjects. Data points marked with an asterisk were included in the
calculation of t1/2 max-12h that describes the decline in plasma concentration up to 12 hours.
Data points marked with a plus symbol were included in the calculation of terminal t1/2. The
6
Pharmacology of LSD
Dolder et al. supplementary material
dotted line represents the respective first-order rate constant. The horizontal dashed line
indicates the lower limit of quantification (0.1 ng/mL).
7
Pharmacology of LSD
Dolder et al. supplementary material
1
S 1 , m a le
*
S 2 , m a le
 = 0 .0 7
*
*
 = 0 .1 2
t 1 / 2 = 9 .4
*
*
0 .1
O -H - L S D (n g /m l)
O -H - L S D (n g /m l)
1
0 .0 1
t 1 / 2 = 5 .7
*
*
*
*
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
16
24
0 1 2 3 4
6
8
10
tim e (h )
1
*
t 1 / 2 = 9 .4
*
*
0 .1
O -H - L S D (n g /m l)
O -H - L S D (n g /m l)
*
0 .0 1
S 8 , m a le
*
 = 0 .1 5
*
t 1 / 2 = 4 .7
*
*
0 .1
6
8
10
12
16
24
0 1 2 3 4
6
8
10
tim e (h )
12
16
24
tim e (h )
1
1
S 9 , fe m a le
t1 / 2 = N D
0 .1
O -H - L S D (n g /m l)
 = ND
O -H - L S D (n g /m l)
24
0 .0 1
0 1 2 3 4
0 .0 1
S 1 3 , m a le
*
 = 0 .1 4
*
t 1 / 2 = 5 .0
*
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
16
24
0 1 2 3 4
6
8
10
tim e (h )
16
1
S 1 5 , f e m a le
24
S 1 6 , f e m a le
 = 0 .1 3
*
 = 0 .0 5
t 1 / 2 = 5 .2
*
*
0 .1
0 .0 1
O -H - L S D (n g /m l)
*
12
tim e (h )
1
O -H - L S D (n g /m l)
16
1
S 3 , fe m a le
 = 0 .0 7
*
12
tim e (h )
*
*
t1 / 2 = 1 4
*
*
*
*
0 .1
0 .0 1
0 1 2 3 4
6
8
10
12
tim e (h )
16
24
0 1 2 3 4
6
8
10
12
16
24
tim e (h )
Supplementary Figure S2. Semilogarithmic plots of the individual 2-oxo-3-hydroxy-LSD
(O-H-LSD) plasma concentration-time profiles of the 8 subjects where O-H-LSD could be
measured in the plasma. Each panel represents data from 1 subject. Filled circles were used
for male subjects and open circles for female subjects. Data points marked with an asterisk
were included in the calculation of t1/2 (dotted line: linear fit through points). The horizontal
dashed line indicates the lower limit of quantification (0.1 ng/mL).
8
Pharmacology of LSD
Dolder et al. supplementary material
9