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4 High-sensitivity and simultaneous analysis of Psychoactive drugs using LC-MS/MS with full-automated pretreatment system Figure 5 Flow diagram of trapping system MS Mobile phase for trapping Mobile phase A+B Drain Trap …
3
High-sensitivity and simultaneous analysis of Psychoactive drugs
using LC-MS/MS with full-automated pretreatment system
Figure 2 CLAM-2000 and LCMS-8060 system
Methods and Materials
The analysis of 60 psychoactive drugs (eight Barbiturate
drug, thirty-nine Benzodiazepines and their metabolites,
twelve Tri-/Tetra- cyclic antidepressant and
bromovalerylurea) were performed using a fully automatic
LCMS preparation unit (CLAM-2000, Shimadzu) online
with HPLC-LCMS (NexeraX2-LCMS-8060, Shimadzu).
Samples were trapped on Imtakt Unison UK-C18
(10x2mm, 3.0μm), then separated by Imtakt Unison
UK-C18 (75x2mm, 3.0μm) with a binary gradient system.
Water with ammonium formate and methanol were used
for mobile phases.
-Serum
-Whole Blood
-Urine
Report creation
MS/MS
Library
search
Filtration
(PTFE,
0.45μm)
• 90sec
Shaking
• 30sec
Filtration
(PTFE,
0.45μm)
• 90sec
Shaking
• 60sec
To AutoSampler
Figure 3 Analytical ow of serum and whole blood
Reagent
Dispensing
• 50 µL of Water
Reagent
Dispensing
• 300 µL of MeOH
Sample
Dispensing
• 50 µL
of serum or whole blood
Shaking
• 60sec
To AutoSampler
Figure 4 Analytical ow of urine
Reagent
Dispensing
• 100 µL of MeOH
Sample
Dispensing
• 100 µL
of urine
4
High-sensitivity and simultaneous analysis of Psychoactive drugs
using LC-MS/MS with full-automated pretreatment system
Figure 5 Flow diagram of trapping system
MS
Mobile phase
for trapping
Mobile phase A+B
Drain
Trap column
Analytical column
Position 0
MS
Mobile phase
for trapping
Mobile phase A+B
Drain
Position 1
Ionization : ESI, Positive/Negative MRM mode
Trap column : Unison UK-C18 (10×2 mm, 3 μm, Imtakt)
Analytical column : Unison UK-C18 (75×2 mm, 3 μm, Imtakt)
Mobile phase for traping : 5% MeOH / 0.1% Formic acid
Mobile phase A : 10mM Ammonium formate, 5% Methanol
B : 10mM Ammonium formate, 95% Methanol
Time program : B conc. 0 % - (1 min) - 5 % - (7 min) - 95 % (3 min)
LC/MS/MS conditions (Nexera system and LCMS-8060)
Usually LC-MS/MS analysis of biological samples require
some manual preparation steps such as protein
precipitation, solid phase extraction or liquid/liquid
extraction before the injection. With the aim to reduce
the operator involvement, to increase the throughput
and the data quality, we completely eliminated the
manual sample preparation procedure by the use of a
novel automatic preparation unit including precipitation,
ltration, incubation, shaking and pipetting. Serum and
whole blood spiked with sixty psychoactive drugs were
pretreated with organic solvent and ltration by the
unit. On the other hands, urine spiked with their drugs
were only ltration. The treated samples were trapped
for cleaning and concentration, then separated by
Unison UK-C18 in HPLC Unit.
The recovery of whole blood spiked with sixty
psychoactive drugs were more than 70% and the
recovery of serum and urine spiked with them were
more than 80%. We completed analysis of their
psychoactive drugs in several biological matrices using
the automated sample preparation system coupled to
LC-MS/MS
Recovery rate
Result
5
High-sensitivity and simultaneous analysis of Psychoactive drugs
using LC-MS/MS with full-automated pretreatment system
Concentration in sample
(μg/mL)
84%
86%
82%
78%
83%
92%
83%
84%
84%
1
80%
80%
91%
80%
92%
80%
84%
81%
84%
10
81%
81%
84%
77%
77%
69%
76%
75%
71%
0.1
Serum
77%
79%
84%
80%
74%
73%
79%
75%
79%
1
76%
78%
79%
76%
81%
77%
77%
77%
79%
10
67%
78%
73%
72%
70%
67%
72%
68%
75%
0.1
Whole blood
103%
103%
102%
109%
101%
107%
97%
110%
104%
1
89%
92%
89%
92%
86%
88%
84%
95%
92%
10
90%
88%
87%
95%
92%
78%
76%
81%
91%
0.1
Urine
Allobarbital (neg)
Amobarbital (neg)
Barbital (neg)
Pentobarbital (neg)
Phenobarbital (neg)
Secobarbital (neg)
Thiamylal (neg)
Thiopental (neg)
Bromovalerylurea
Concentration in sample
(μg/mL)
69%
73%
70%
72%
68%
70%
68%
84%
73%
69%
75%
0.1
80%
80%
74%
77%
80%
81%
76%
76%
80%
80%
83%
1
74%
72%
67%
74%
70%
76%
68%
79%
71%
71%
83%
0.01
Serum
66%
66%
65%
60%
64%
67%
58%
77%
64%
104%
70%
0.1
70%
73%
83%
89%
77%
87%
67%
81%
70%
116%
83%
1
64%
66%
63%
57%
63%
64%
56%
78%
55%
89%
68%
0.01
Whole blood
84%
86%
78%
83%
86%
84%
83%
85%
83%
84%
87%
0.1
102%
101%
99%
103%
100%
100%
101%
111%
100%
103%
106%
1
87%
90%
82%
90%
88%
86%
91%
90%
91%
89%
87%
0.01
Urine
Amitriptyline
Amoxapine
Clomipramine
Desipramine
Dosulepin
Imipramine
Maprotiline
Mianserin
Nortriptyline
Promethazine
Setiptiline