An automated method was developed to extract and analyze barbiturates and THCA from human urine using ITSP solid phase extraction and liquid chromatography-mass spectrometry. Standards of six barbiturates and THCA were prepared in urine over ranges of 0.1-20 μg/mL and 0.01-2.0 μg/mL respectively and processed using an ITSP cleanup followed by LC-MS/MS analysis. Linear calibration curves were obtained for all analytes with R2 values greater than 0.99, demonstrating the method is suitable for quantifying the target compounds in urine.

1. Microliter Analytical Supplies, Inc. 1-888-232-7840, INFO@MICROLITER.COM, WWW.MICROLITER.COM
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Determination of Barbiturates and THCA in Human Urine Using Automated ITSP Solid
Phase Extraction and Liquid Chromatography Mass Spectrometric Detection
Thurman L. Allsup1
, Kenneth C. Lewis1
, Rick Youngblood2
, and Kim Gamble2
1
OpAns, LLC, Durham NC, 27713, 2
Microliter Analytical Supplies, Inc., Suwannee GA, 30024
Abstract
A quantitative analytical method has been developed
for the determination of phenobarbital, butabarbital,
butalbital, secobarbital, amobarbital, pentobarbital
and 11-Nor-Δ9-tetrahydrocannabinol-9-carboxylic
acid (THCA) in human urine. The method that has
been developed here is fully automated to
quantitatively extract barbiturates and THCA from
urine using QAX (strong anion exchange) ITSP SPE
devices and analyze the samples in-line by
LC/MS/MS. The sample extraction and injection
onto the LC/MS/MS were both performed in-line by
a PAL (CTC) sample handling unit. Spiked urine
samples with stable label internal standards were
adjusted to basic pH with pH 10.5 buffer and then
applied to QAX ITSP devices for extraction. The
extracts were injected in-line onto an LC/MS/MS
using a XTerra MS C18, 3.5 µm, 2.1x50 mm column
for separation. The extracted standard curve had a
range of 0.1 - 20 µg/mL for the barbiturates and 0.01
– 2 µg/mL for THCA. The correlation coefficients
were greater than 0.99 for all compounds. Selected
reaction monitoring (SRM) was used for the
quantitation.
Butalbital
Exact Mass 224.2
Butabarbital
Exact Mass 212.2
Phenobarbital
Exact Mass 232.2
Secobarbital
Exact Mass 238.3
Pentobarbital
Exact Mass 226.2
Amobarbital
Exact Mass 226.2
THCA
Exact Mass 344.2
Experimental
Sample Preparation
Phenobarbital, butabarbital, butalbital, secobarbital,
pentobarbital, amobarbital, and THCA standards and
the deuterated analogs of butalbital, secobarbital,
phenobarbital, pentobarbital and THCA were
obtained from Cerillant. A set of standards was
prepared in human urine. Calibration standards were
prepared at nominal concentrations of 0.1, 0.5, 1, 10,
and 20 µg/mL with a constant concentration of the
deuterated analog at 1 µg/mL for the barbiturates.
Calibration standards were prepared at nominal
concentrations of 0.01, 0.05, 0.1, 1.0, and 2.0 µg/mL
with a constant concentration of the deuterated
analog at 0.1 µg/mL for THCA. Standards were pH
adjusted with 100 L of pH 10.5 buffer.

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ITSP SPE Method
ITSP Cartridges: UCT SelectrasorbTM
QAX 10mg
(Product No.: 07-UQAX10-20A)
A CTC Analytics HTS PAL sample handler was used
to prepare the samples. The PAL was configured
with a 100 L syringe and two tray holders. Each
tray holder held 2 microplates, one of which was
designed to hold the ITSP hardware kit (Product No.:
07-ITSP-HW). The extraction protocol was as
follows:
Step Solvent Volume
( L)
Flowrate
( L/sec)
Load Sample 500 10
Wash A 100 10
Wash B 100 10
Flush Air 100 100
Elute C 100 5
Flush Air 100 100
Solvent A: 100mM Carbonate pH 10.5 buffer
Solvent B: 1% Ammonium hydroxide in methanol
Solvent C: 5% Acetic acid in methanol
Samples were prepared and analyzed in-line with the
following method:
Analysis Method
Instrument: Finnigan TSQ Quantum Ultra AM triple
quadrupole with Agilent 1200 Rapid
Resolution HPLC
Column: XTerra MS C18, 2.1x50 mm, 3.5 m
particles
Solvent A: Water with 5 mM ammonium acetate
and 0.05% ammonium hydroxide
Solvent B: Methanol with 0.05% ammonium
hydroxide
Column Temp.: Ambient
Flowrate: 0.5 mL/min
Gradient: 0.00 min (10% B), 0.5 min (10% B), 4.0
min (40% A), 5.6 min (100% B), 5.8
min (100% B), 6.0 min (10% B)
Ionization Mode: Negative Ion (HESI) Heated
Electrospray Ionization Probe
SRM Channels:
Quant. Confirm IS
Butabarbital 211-211 211-42 Pheno.
Butalbital 223-223 223-42 228-228
Secobarbital 237-237 237-42 242-242
Phenobarbital 231-231 231-42 236-236
Amobarbital 225-225 225-42 Pento
Pentobarbital 225-225 225-42 230-230
THCA 343-299 343-245 346-302
Results
Standards of the six barbiturates and THCA were
prepared in urine over the range of 0.1 - 20 µg/mL
and 0.01 – 2.0 µg/mL, respectively, and processed
in-line with an ITSP cleanup procedure followed by
LC-MS/MS. The calibration curves that were
obtained are shown below, plotting the analyte to
internal standard area ratio versus the concentration
of the analyte. Because of the barbiturates extremely
poor fragmentation, the precursor to precursor SRM
transition was used for all barbiturates and the
confirmatory SRM transition was only useful for the
higher concentrations.
Butalbital
Y = 0.00935706+0.84444*X R^2 = 0.9994 W: 1/X
0 2 4 6 8 10 12 14 16 18 20 22
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
AreaRatio
Ratio
Phenobarbital
Y = -0.0464945+1.08484*X R^2 = 0.9989 W: 1/X
0 2 4 6 8 10 12 14 16 18 20 22
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
AreaRatio
Ratio

3. Microliter Analytical Supplies, Inc. 1-888-232-7840, INFO@MICROLITER.COM, WWW.MICROLITER.COM
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Conclusions
A simplified, automated procedure for the analysis of
butabarbital, butalbital, secobarbital, phenobarbital,
pentobarbital, amobarbital and THCA in human urine
has been developed using ion exchange ITSP SPE for
in-line sample cleanup and LC-MS/MS for detection.
A linear response was observed over the
concentration range of 0.1 - 20 µg/mL for all of the
barbiturates and over the range of 0.01 - 2 µg/mL for
THCA.
THCA
Y = -0.0222865+7.79162*X R^2 = 0.9945 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
AreaRatio
Ratio
Secobarbital
Y = -0.0401594+0.815851*X R^2 = 0.9906 W: 1/X
0 2 4 6 8 10 12 14 16 18 20 22
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
AreaRatio
Ratio
Pentobarbital
Y = -0.0418638+0.883712*X R^2 = 0.9919 W: 1/X
0 2 4 6 8 10 12 14 16 18 20 22
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
AreaRatio
Ratio
Amobarbital
Y = 0.0936486+0.810262*X R^2 = 0.9913 W: 1/X
0 2 4 6 8 10 12 14 16 18 20 22
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
AreaRatio
Ratio
Butabarbital
Y = -0.00965978+0.953161*X R^2 = 0.9997 W: 1/X
0 2 4 6 8 10 12 14 16 18 20 22
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
AreaRatio
Ratio