Journal of Analytical Toxicology, Vol. 33, June 2009
Validated Ultra-Performance Liquid Chromatography–
Tandem Mass Spectrometry Method for Analyzing
LSD, iso-LSD, nor-LSD, and O-H-LSD in
Blood and Urine
Angela Chung1,2, John Hudson3, and Gordon McKay4
1University
of Saskatchewan, College of Graduate Studies and Research, Toxicology Graduate Program, Saskatoon,
Saskatchewan, Canada; 2Royal Canadian Mounted Police, Forensic Science and Identification Services, Winnipeg, Manitoba,
Canada; 3Royal Canadian Mounted Police, Forensic Science and Identification Services, Regina, Saskatchewan, Canada; and
4University of Saskatchewan, College of Pharmacy and Nutrition, Saskatoon, Saskatchewan, Canada
Abstract
The Royal Canadian Mounted Police Forensic Science and
Identification Services was looking for a confirmatory method for
lysergic acid diethylamide (LSD). As a result, an ultra-performance
liquid chromatography–tandem mass spectrometry method was
validated for the confirmation and quantitation of LSD, iso-LSD,
N-demethyl-LSD (nor-LSD), and 2-oxo-3-hydroxy-LSD (O-H-LSD).
Relative retention time and ion ratios were used as identification
parameters. Limits of detection (LOD) in blood were 5 pg/mL for
LSD and iso-LSD and 10 pg/mL for nor-LSD and O-H-LSD. In
urine, the LOD was 10 pg/mL for all analytes. Limits of
quantitation (LOQ) in blood and urine were 20 pg/mL for LSD and
iso-LSD and 50 pg/mL for nor-LSD and O-H-LSD. The method was
linear, accurate, and precise from 10 to 2000 pg/mL in blood and
20 to 2000 pg/mL in urine for LSD and iso-LSD and from 20 to
2000 pg/mL in blood and 50 to 2000 pg/mL in urine for nor-LSD
and O-H-LSD with a coefficient of determination (R2) ≥ 0.99. The
method was applied to blinded biological control samples and
biological samples taken from a suspected LSD user. This is the
first reported detection of O-H-LSD in blood from a suspected
LSD user.
Introduction
The analysis of lysergic acid diethylamide (LSD) in biological
fluids has been a challenge. LSD is generally taken in small
doses (1), is metabolized rapidly (2), has a short half-life (3),
and is an unstable compound (4). The biggest challenge analytically is related to the low concentration of LSD and its
metabolites in biological fluids. Therefore, a highly sensitive
method is needed to detect and confirm the presence of LSD
and its metabolites. To increase the window of detection for
LSD use, recent methods have been targeting the metabolites
of LSD because they have longer half-lives and are present in
higher concentrations than LSD. The half-life of N-demethylLSD (nor-LSD) is 10 h (5), and LSD has a half-life of 5.1 h (6).
More recently, 2-oxo-3-hydroxy-LSD (O-H-LSD) was determined to be a major metabolite of LSD (7), and it has been reported in concentrations of up to 25 times higher than LSD in
the urine (8).
Additionally, previous methods have also targeted the isomer
of LSD. The diastereoisomer impurity (iso-LSD) is formed
during the production of illicit LSD from lysergic acid (9).
iso-LSD can also form when LSD is exposed to basic aqueous
solutions and increased temperatures (4,10), ultimately
reaching an LSD/iso-LSD ratio of 9:1 in the sample (10,11).
Iso-LSD, nor-LSD, and O-H-LSD do not have any psychoactive
properties, but their presence in biological fluids is an indication of LSD use.
A number of gas chromatography–mass spectrometry
(GC–MS) methods have been developed to identify LSD
(12–15). However, the detection of LSD using GC–MS has its
challenges. LSD undergoes irreversible adsorption during the
chromatographic process, is relatively nonvolatile, and is not
stable at the elevated temperatures associated with GC (12).
Furthermore, sample preparation is laborious, requiring a
derivatization step (12,16). Because of the difficulties in detecting LSD by GC–MS and the increase in popularity of liquid
chromatography (LC), recent identification methods developed for LSD analysis have used LC–MS (8,16–23) or
LC–MS–MS (9,23–27).
The Royal Canadian Mounted Police (RCMP) Forensic Science
and Identification Services (FS&IS) has been involved in the
identification of LSD in biological samples for the past 15 years.
An enzyme-linked immunosorbant assay (ELISA) from Immunalysis® (Pomona, CA) is currently being used by the RCMP
FS&IS as a rapid screening method for LSD. In 1997, the RCMP
FS&IS developed a confirmatory GC–MS method using the
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253
Journal of Analytical Toxicology, Vol. 33, June 2009
Zymark Rapid Trace solid-phase extractor for the trimethylsilyl
derivative of LSD, with a limit of quantitation (LOQ) of 100
pg/mL. The method was shown to be useful for case samples, but
had its disadvantages: 1. it could not detect LSD metabolites, 2.
it required large sample volumes (4 mL blood or 5 mL urine), 3.
a derivatization step was necessary, and 4. the GC required preconditioning of the column to prevent the adsorption of LSD.
As a result, LC has been employed as an alternative to GC.
This paper describes an ultra-performance liquid chromatography (UPLC)–MS–MS method for the analysis of LSD, isoLSD, nor-LSD, and O-H-LSD in blood and urine samples.
Linearity, accuracy, precision, sensitivity, selectivity, recovery,
matrix effects, and reproducibility were evaluated in the validation. The method was applied to spiked human blood and
urine samples blinded to the analysts, as well as biological
samples from a suspected LSD user.
Materials and Methods
Chemicals and reagents
LSD, iso-LSD, nor-LSD, O-H-LSD, and deuterated LSD
(LSD-d3) were obtained from Cerilliant (Round Rock, TX). All
reagents were high-performance liquid chromatography
(HPLC) or reagent grade. Acetonitrile (ACN), ammonium acetate, ammonium hydroxide, and methylene chloride were obtained from Fisher Scientific (Fairlawn, NJ). Isopropyl alcohol
and glacial acetic acid were obtained from Caledon Laboratories
(Georgetown, ON, Canada). All water was purified with the
NANOpure II water purification system (Barnstead, a division of
Apogent Technologies, Dubuque, IA). Synthetic drug-free urine
was obtained from Immunalysis. Drug-free porcine blood was
obtained from Maple Leaf Meats (Winnipeg, MB, Canada) (diluted 25% with water, and sodium fluoride and potassium oxalate were added to concentrations of 0.25% and 0.20%,
respectively). Drug-free blood samples were collected from
human volunteers into BD Vacutainers® (XF947).
Standard solutions
Individual stock solutions of LSD, iso-LSD, nor-LSD, and
O-H-LSD, each at a concentration of 500 ng/mL, were prepared in ACN. Using the stock solutions, two working solutions
(1 and 20 ng/mL) containing all analytes (i.e., LSD, iso-LSD,
nor-LSD, and O-H-LSD) were prepared in ACN. The working
solutions were then used to prepare nine calibrators (5, 10,
20, 50, 100, 400, 800, 1600, and 2000 pg/mL). Each calibrator
contained all four analytes spiked into drug-free porcine blood
or synthetic urine. The working solutions were also used to prepare control samples (50, 500, and 1500 pg/mL) containing all
analytes spiked into drug-free porcine blood or synthetic urine.
A 500 ng/mL internal standard (ISTD) (LSD-d3) stock solution
was prepared in ACN and further diluted with ACN to prepare
a 20 ng/mL internal standard working solution. LSD-d3 was
used as the internal standard for all analytes.
LC conditions
A Waters (Milford, MA) Acquity UPLC with an autosampler
using an Acquity UPLC Bridged Ethyl Hybrid (BEH) C18 (2.5
254
× 50 mm, 1.7 µm, Waters) column was used for chromatographic separation. Separation was conducted at 30°C. A gradient elution was conducted using 20 mM ammonium acetate
buffer (pH 4.0) and ACN at a flow rate of 0.2 mL/min. A gradient of buffer/ACN (90:10) was held for 1 min, followed by a
linear step gradient to buffer/ACN (75:25) for 10 min, and held
for 1 min. The column was then equilibrated for 2 min, giving
a total run time of 15 min.
MS conditions
A Waters Quattro Premier™ tandem-quadrupole MS
equipped with a Z-spray™ ion source was coupled to the UPLC
system. Atmospheric pressure ionization (API) was done via
positive electrospray ionization (ESI). The electrospray probe
tip potential was set at 3.00 kV. The source and desolvation
temperatures were set at 120 and 350°C, respectively. The
cone and desolvation gas flows were set at 110 and 747 L/h, respectively. The collision gas (argon) flow and pressure were set
at 0.28 mL/min and 4.21 × 10–3 mbar, respectively. The MS was
run in multiple reaction monitoring (MRM) mode with a dwell
time of 0.20 s (interscan delay of 0.01 s) (Table I).
Sample preparation
Sample preparation procedure was adapted from previously
published methods (21,27). To 1 mL of each sample, 50 µL of
LSD-d3 ISTD working solution (20 ng/mL) was added, and the
sample was vortex mixed for 10 s. Then 500 µL of 1 M ammonium acetate buffer (pH 9.0) was added, and the sample was
vortex mixed again for 10 s. Each sample was extracted with
5 mL of dichloromethane/isopropyl alcohol (85:15). The tube
was capped, manually shaken for 5 s, and then placed on a flatbed shaker (Eberbach, Ann Arbor, MI) at low speed for 20 min.
Each tube was centrifuged at 3500 rpm for 15 min. The
aqueous (top) layer was discarded. The organic (bottom) layer
was transferred to a clean tube and evaporated to dryness (N2
stream, room temperature). The residue was reconstituted in
100 µL of 20 mM ammonium acetate buffer (pH 4.0)/ACN
(80:20), and 20 µL of sample was injected onto the UPLC
system.
Validation procedure
Three sets of calibrators (including blanks) were prepared
fresh daily and extracted as described here previously, on three
Table I. UPLC–MS–MS MRM Parameters
Standard
Precursor Ion
[M+H]+
LSD
324
iso-LSD
nor-LSD
O-H-LSD
LSD-d3
324
310
356
327
Product Ion
Collision
Cone
Voltage
223*
208
281
281*, 208, 223
209*, 237, 74
237*, 222, 313
226*, 208, 281
24
31
19
24
24
25
24
35
35
35
35
37
35
35
* Product ions to be used for quantitation; other ions are qualifier ions.
Journal of Analytical Toxicology, Vol. 33, June 2009
separate days. Data collection, peak integration, and weighted
(1/x2) linear regression were performed using MassLynx™
version 4.0 software (Waters). Nominal concentration versus
peak-area ratio values were plotted to define the calibration
curve. A suitable linear range was determined by evaluating the
accuracy (% bias) of calculated results.
Six sets of controls (50, 500, and 1500 pg/mL) were prepared
fresh daily and extracted to assess intraday accuracy and precision. This was then repeated on three separate days to assess
interday accuracy and precision. Control results were calculated from the equation as determined by the weighted (1/x2)
linear regression of the calibration curve. The accuracy of the
method was determined by % bias. The precision of the
method was determined by the coefficient of variation (%CV).
Limits of detection (LOD) and quantitation (LOQ) were assessed by determining the peak-to-peak signal-to-noise ratio
(S/N) using the MassLynx™ version 4.0 software. LOQ must
also demonstrate acceptable accuracy and precision. Another
parameter assessed was selectivity. Six different sources of
drug-free urine and blood matrix were extracted and analyzed.
Each source of drug-free urine and blood was then tested for
interference at the LOQ.
The percentage recovery was assessed in two matrices (i.e.,
porcine blood and synthetic urine) by preparing two sets of
samples in each matrix. Each set contained all analytes and was
prepared in replicates of six at 50 and 1500 pg/mL. Samples in
the first set were prepared by adding the appropriate volume of
working solution to drug-free matrix, and then the samples
were extracted. Samples in the second set were prepared by extracting drug-free matrix then adding the appropriate volume
of working solution to the extracts. The peak-area ratios of
samples in the first set were compared to the samples in the
second set to determine percentage recovery.
Matrix effect was assessed by using a previously published experimental procedure (28). Five different lot numbers of drugfree synthetic urine and one drug-free human urine sample
were evaluated. Drug-free porcine blood and drug-free human
blood (five sources) were also evaluated.
For further confirmation of the analytes, other factors such
as relative retention time (RRT) and ion ratios were included.
The ion ratio was derived from the peak area of the product ion
in one MRM divided by the peak area of another product ion in
a second MRM.
Results and Discussion
Optimization of the MS conditions was done by direct infusion of the drug standards (2 µg/mL) at 20 µL/min through a
syringe pump into a T piece with mobile phase. MS parameters
were adjusted to maximize sensitivity of product ions produced by collision-induced dissociation of protonated ions.
LSD, iso-LSD, and nor-LSD, and 20 to 2000 pg/mL for O-HLSD. The coefficient of determination (R2) of all analytes was
greater than 0.99 in both urine and blood. The calibration
curves and correlation coefficients for all analytes are as follows: y = 0.00101 (SE 0.00002) x + 0.0049 (SE 0.0009), r =
0.997 (LSD in urine); y = 0.00107 (SE 0.00002) x + 0.0035 (SE
0.0006), r = 0.999 (LSD in blood); y = 0.00110 (SE 0.00002) x
+ 0.0082 (SE 0.0010), r = 0.998 (iso-LSD in urine); y = 0.00089
(SE 0.00001) x – 0.0016 (SE 0.0007), r = 0.999 (iso-LSD in
blood); y = 0.00047 (SE 0.00001) x + 0.036 (SE 0.0008), r =
0.996 (nor-LSD in urine); y = 0.00052 (SE 0.00001) x +
0.00086 (SE 0.0003), r = 0.998 (nor-LSD in blood); y = 0.00026
(SE 0.00001) x + 0.0027 (SE 0.0007), r = 0.993 (O-H-LSD in
urine); and y = 0.00031 (SE 0.00001) x – 0.0003 (SE 0.0003),
r = 0.998 (O-H-LSD in blood).
Accuracy and precision
The accuracy was deemed acceptable if the % bias of the calculated results of the control samples were ± 15% and ± 20%
at LOQ of the nominal concentration. The precision was
deemed acceptable if the %CV of the calculated results of the
control samples were less than 15% and 20% at LOQ. Quantitations are done mainly on blood samples. Urine samples are
used for qualitative analysis. As a result, for the validation study
quantitative analysis for blood was conducted using a full calibration curve with six points, whereas semi-quantitative analysis for urine was conducted using a three-point calibration
curve. Both inter- and intraday accuracy and precision for all
analytes in both urine and blood were within acceptable limits.
The accuracy and precision for all analytes in both blood and
urine ranged from –13 to 4% bias and 3 to 14% CV, respectively.
LOD and LOQ
LOD and LOQ are defined as where the S/N is greater than
3:1 and 10:1, respectively. In addition to a S/N greater than
10:1, the LOQ must also show acceptable accuracy (± 20%
bias) and precision (< 20% CV). In urine, the LOD was 10
pg/mL for all analytes, and the LOQ was 20 pg/mL for LSD and
iso-LSD and 50 pg/mL for nor-LSD and O-H-LSD. In blood, the
LOD was 5 pg/mL for LSD and iso-LSD and 10 pg/mL for norLSD and O-H-LSD, and the LOQ was 20 pg/mL for LSD and
iso-LSD and 50 pg/mL for nor-LSD and O-H-LSD. LOD and
LOQ values in this study showed very good sensitivity, and
this can most likely be attributed to the highly efficient UPLC
columns. Chromatography can affect the LOD and LOQ
wherein better peak shape gives a higher S/N ratio, resulting
in a lower LOD and LOQ (29).
Selectivity
The Food and Drug Administration (FDA) recommends that
the LOQ have at least five times the response compared to
the blank (30). Six different sources of blank urine and blood
were extracted and analyzed, and no interferences were found
at the LOQ.
Linearity and range
The linear range in urine was 20 to 2000 pg/mL for both
LSD and iso-LSD and 50 to 2000 pg/mL for both nor-LSD and
O-H-LSD. The linear range in blood was 10 to 2000 pg/mL for
Recovery
Percentage recoveries of LSD and related analytes ranged
from 74 to 88% in urine and 61 to 69% in blood. The recov-
255
Journal of Analytical Toxicology, Vol. 33, June 2009
eries found in this study were similar to the liquid–liquid extraction (LLE) methods reported in the literature (21,27). The
percentage recoveries were reproducible with a %CV range of
3.9 to 11.4% in urine and 2.9 to 8.1% in blood for all analytes.
Both high (1500 pg/mL) and low (50 pg/mL) concentrations of
each analyte also showed similar percentage recoveries.
Matrix effect
Both urine and blood demonstrated similar absolute matrix effect at both the high (1500 pg/mL) and low (50 pg/mL)
concentrations. LSD, iso-LSD, O-H-LSD, and LSD-d3 showed
slight ion suppression, whereas nor-LSD showed slight ion
enhancement. One ion suppression study done by Johansen
and Jensen (9) was conducted by injecting extracted spiked
Table II. Results of the Control Samples Blinded to Two
Different Analysts
Sample
Analyte
Spiked
Level
(pg/mL)
porcine blood extracts through a syringe pump into a T piece
with mobile phase. Authors in that study found no evidence of
ion suppression for LSD and iso-LSD and stated that the
deuterated ISTD corrected for any possible suppression of ions.
Even though the absolute matrix effect of nor-LSD was different compared to the other analytes, this does not necessarily
indicate that the bioanalytical method was invalid. If the relative matrix effect exhibits the same pattern for the analyte and
the ISTD in all sources of matrix studied, the analyte to ISTD
ratio should not be affected (28). The relative matrix effect, defined as the comparison of matrix effect values between different sources of the matrix, showed no significant difference
(p > 0.05, F test) between the variances of the peak-area ratio
of neat standards in ACN versus blood and urine. The relative
matrix effect of all analytes was not shown to differ significantly, confirming that the matrix effect had no effect on the
quantitation of LSD, iso-LSD, nor-LSD, and O-H-LSD.
Other qualitative parameters
Measured Values (pg/mL)
Analyst 1
Analyst 2
Urine #1 LSD
iso-LSD
nor-LSD
O-H-LSD
3000
2400
0
8800
> 1500 (2549*)
> 1500 (2424*)
ND†
> 1500 (9089*)
> 1500 (2484*)
> 1500 (2281*)
ND
> 1500 (9642*)
Urine #2 LSD
iso-LSD
nor-LSD
O-H-LSD
100
600
100
6500
89
578
88
> 1500 (6765*)
88
566
83
> 1500 (6950*)
ND
ND
ND
> 1500 (9014*)
ND
ND
ND
> 1500 (9012*)
The RRTs of all analytes were precise (%CV ≤ 1.7%) over
three days of analysis in both urine and blood. The RRT was
deemed acceptable as a parameter of confirmation if the control samples did not differ by more than 2% (% bias) relative to
the calibrators (31).
Table III. Case Sample Results
% Bias of Case Sample
Relative to the Calibrators
RRT
Ion
ratio 1
Ion
ratio 2
Concentration
(pg/mL)
Femoral blood
LSD
iso-LSD
nor-LSD
O-H-LSD
–0.2
–0.2
–0.1
–0.8
0.00
11.4
–49.3†
–1.5
–9.4
7.2
–9.4
14.1
561
Trace*
ND‡
117
Subclavian blood
LSD
iso-LSD
nor-LSD
O-H-LSD
0.3
0.1
0.1
–0.3
–7.3
5.2
–9.2
4.0
–10.9
3.9
32.1†
–3.0
536
Trace
ND
166
Analyte
Urine #3 LSD
iso-LSD
nor-LSD
O-H-LSD
0
0
0
8000
Urine #4 LSD
iso-LSD
nor-LSD
O-H-LSD
0
500
0
10,000
Blood #1 LSD
iso-LSD
nor-LSD
O-H-LSD
270
440
0
1500
219
394
ND
1295
155
338
ND
1298
Blood #2 LSD
iso-LSD
nor-LSD
O-H-LSD
1000
750
200
1000
774
694
139
924
563
615
121
889
LSD
iso-LSD
nor-LSD
O-H-LSD
–0.6
–0.4
–0.4
0.2
–3.6
9.1
3.1
–4.5
–11.1
5.1
–6.6
17.2
> 1500§
73
188
> 1500#
Blood #3 LSD
iso-LSD
nor-LSD
O-H-LSD
0
500
0
1000
ND
476
ND
919
ND
454
ND
991
Blood #4 LSD
iso-LSD
nor-LSD
O-H-LSD
0
0
0
500
ND
ND
ND
450
ND
ND
ND
405
Vitreous humor**
LSD
iso-LSD
nor-LSD
O-H-LSD
–0.3
0.1
–50.1†
8.2†
4.2
160.8†
–91.6†
–62.2†
–9.1
–96.7†
–100.0†
–67.9†
74
ND
ND
ND
ND
ND
489
482
ND
ND
> 1500 (10,632*) > 1500 (10,529*)
* Result from extrapolating the calibration curve above the highest calibrator.
† ND = none detected.
256
Urine
* Trace = result ≥ LOD but < LOQ.
† Outside acceptable parameters for confirmation. Acceptable parameters are
± 2% bias for RRT and ± 30% bias for ion ratios.
‡ ND = none detected.
§ Extrapolated results = 2332 pg/mL.
# Extrapolated results = 9286 pg/mL.
** The method has not been validated for vitreous humor sample matrix.
Journal of Analytical Toxicology, Vol. 33, June 2009
MRM ion ratios of the calibrators were found to be precise,
and the ion ratios of the control samples were found to be accurate (± 30% bias) relative to the mean ion ratio of the calibrators. The ion ratios were also found to be concentration
dependent. The precision at the low (50 pg/mL) concentration
was less than at the higher (1500 pg/mL) concentration, even
more so for O-H-LSD. Because it was demonstrated that the
ion ratios were concentration dependent, a ± 30% bias was
chosen for the acceptance criteria (32).
Control samples blinded to the analysts
To test the reliability of this method, eight control samples
(four urine and four blood) were prepared by another laboratory using drug-free human urine and blood (McKay, Pharmalytics, Saskatoon, SK, Canada). The actual concentrations in
these control samples were blinded to the analysts. Each control sample was extracted in
duplicate. Analysis was conducted by two different analysts on two different days. The results obtained with the UPLC–MS–MS method
are summarized in Table II.
tracted in duplicate and analyzed using the method described
earlier. The results obtained in the case samples are summarized in Figure 1 and Table III. Even 10 years after collection,
LSD and metabolites were still detectable. However, because no
quantitation was conducted on the samples 10 years ago, the
stability of the analytes cannot be assessed. Nonetheless, the results show that LSD and metabolites can still be detected if kept
in a frozen state.
To our knowledge, this is the first time that O-H-LSD has
been detected and confirmed in blood. The majority of the
methods published for O-H-LSD have only attempted analysis
in urine (8,23,24). Methods that have attempted to confirm
LSD and metabolites, including O-H-LSD, in blood were unable to detect O-H-LSD even when the blood was positive for
LSD (21,27). Previous LC–MS and LC–MS–MS methods may
Case study
This validated method was used to analyze
samples from a 1995 case that was found to be
positive for LSD using radioimmunoassay
(RIA). The scenario provided was as follows: a
19-year-old male leaped out of a 16th floor
window and landed on the 5th floor of a
parking garage. The ultimate cause of death
was “multiple blunt force injuries”. His friends
said that he might have taken LSD. When the
father was questioned, he indicated that he
was not aware of any specific history of drug
abuse, depression, or thoughts of suicide by
his son. At autopsy, four samples were collected: 1. femoral blood collected in a grey
stopper (potassium oxalate, sodium fluoride)
Vacutainer, 2. subclavian blood collected in a
plastic screw-top container, 3. vitreous humor
collected in a Vacutainer with no additives,
and 4. urine collected in a grey-stoppered
(potassium oxalate, sodium fluoride) Vacutainer. In 1995, an alcohol and routine toxicology screen (immunoassay and GC–MS)
were conducted. No alcohol or other common
drugs were identified in the routine screen except for 3 mg/L acetaminophen in the blood.
Based on the history, an LSD RIA kit was obtained and run with appropriate controls. All
samples “screened positive”. The final 1995
toxicology report noted a “presumptive” LSD
result but that no confirmatory method was
available. After the analysis was completed in
1995, the samples were refrigerated for about
2–4 months and then kept frozen in a –50°C
freezer for 10 years.
In December 2005, all samples were ex-
Time (min)
Figure 1. MRM acquisitions of case sample (femoral blood) for LSD-d3 (A), LSD (561 pg/mL) (B),
iso-LSD (trace) (C), nor-LSD (not detected) (D), and O-H-LSD (117 pg/mL) (E).
257
Journal of Analytical Toxicology, Vol. 33, June 2009
have been unsuccessful in detecting O-H-LSD in blood because of a lack of sensitivity in the extraction. One method
using an Agilent HPLC coupled to a mass selective detector was
unable to detect O-H-LSD in blood (21). When blood spiked
with O-H-LSD was extracted, percentage recovery was 32 to
42%, compared to 69 to 73% in urine. The low recovery in
blood is likely due to the solid-phase extraction step done after
the initial LLE. The LOD and LOQ (400 pg/mL) were only determined for urine. Nonetheless, an LOD and LOQ of 400
pg/mL for O-H-LSD would not have been sufficiently sensitive
to detect O-H-LSD in this case sample.
Like the blind control samples, several of the results for
LSD and O-H-LSD from the case sample were reported as >
1500 pg/mL because the concentration was higher than the
highest calibrator (1500 pg/mL). Extrapolating the calibration
curve showed that O-H-LSD was present in urine at higher
concentrations than LSD. Others have found that the concentration of O-H-LSD is 16 to 25 times higher than that of LSD
(8,23).
This method was not validated for a vitreous humor matrix.
Because it was shown that vitreous humor samples may also
contain LSD, validating the method for vitreous humor could
be done in future experiments. Favretto et al. (33) validated an
LC–MS–MS (LCQ Duo ion trap MS) method for LSD, iso-LSD,
nor-LSD, and O-H-LSD in vitreous humor. They reported an
LOD and LOQ of 10 and 20 pg/mL, respectively, for LSD and
nor-LSD. That method was also used for a vitreous humor
case sample. Results were 2.9 ng/mL LSD and 2.2 ng/mL norLSD.
Conclusions
An accurate, precise, selective, and sensitive UPLC–MS–MS
method was validated to identify, confirm, and quantitate LSD,
iso-LSD, nor-LSD, and O-H-LSD in both urine and blood.
Compared with the GC–MS method previously utilized by the
RCMP since 1997, the UPLC–MS–MS method was more suitable for the analysis of LSD and metabolites in blood and urine,
providing better sensitivity and greater ease of analysis while
using a smaller sample volume.
To the best of our knowledge, there are no published validated methods for the simultaneous confirmatory and quantitative analysis of LSD, iso-LSD, nor-LSD, and O-H-LSD in
urine and blood. Furthermore, compared with other published
methods, the LOD and LOQ obtained in this study for O-H-LSD
and nor-LSD for both blood and urine are among the lowest. In
conclusion, a validated UPLC–MS–MS method is now available
for confirming the use of LSD in blood and urine.
Acknowledgments
Thanks to both the departments of Toxicology and Pharmacy & Nutrition at the University of Saskatchewan and to the
RCMP FS&IS for financial support and the use of equipment
258
and facilities. Special thanks to my supervisors, John Hudson
(RCMP FS&IS Regina) and Dr. Gordon McKay (University of
Saskatchewan, College of Pharmacy & Nutrition) and to the
other members of my committee, Dr. Barry Blakley (University
of Saskatchewan, Toxicology Coordinator) and Dr. Andrew Ross
(National Research Council Canada, Plant Biotechnology Institute) for their constant support and advice. Thanks to the
staff at the RCMP FS&IS Winnipeg for technical and laboratory
assistance, donating blood samples, and for helpful discussions and suggestions, to Cameron Lyttle for analyzing the
blind control samples, and to Dr. Graham Jones (Alberta Medical Examiner) for providing the case samples.
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Manuscript received November 5, 2008;
revision received February 2, 2009.
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