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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% …
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
6
High-sensitivity and simultaneous analysis of Psychoactive drugs
using LC-MS/MS with full-automated pretreatment system
Concentration in sample
(μg/mL)
70%
74%
72%
73%
70%
73%
79%
77%
73%
72%
73%
76%
74%
73%
71%
79%
64%
73%
73%
70%
77%
70%
73%
73%
73%
82%
78%
76%
71%
70%
72%
70%
74%
70%
72%
74%
71%
72%
71%
0.1
82%
78%
86%
81%
80%
74%
82%
77%
79%
81%
80%
79%
80%
93%
79%
73%
87%
76%
78%
79%
73%
80%
81%
77%
89%
73%
70%
82%
82%
83%
87%
76%
79%
79%
78%
78%
78%
78%
86%
1
69%
73%
72%
76%
84%
73%
90%
82%
72%
82%
77%
79%
79%
75%
75%
82%
56%
76%
75%
80%
81%
74%
75%
80%
N.A.
78%
73%
78%
73%
72%
71%
84%
70%
79%
75%
75%
74%
74%
74%
0.01
Serum
75%
76%
73%
73%
77%
76%
80%
74%
74%
73%
75%
74%
73%
73%
70%
77%
68%
70%
73%
76%
75%
72%
75%
74%
73%
78%
77%
73%
75%
76%
74%
72%
70%
70%
75%
75%
73%
75%
74%
0.1
86%
77%
85%
82%
90%
85%
89%
83%
82%
80%
95%
87%
82%
97%
81%
92%
75%
76%
79%
91%
103%
84%
85%
77%
88%
86%
81%
70%
85%
85%
95%
77%
76%
80%
79%
80%
88%
79%
95%
1
78%
82%
79%
77%
78%
81%
81%
80%
80%
76%
81%
74%
76%
73%
70%
79%
55%
86%
75%
77%
83%
79%
76%
75%
N.A.
81%
81%
71%
78%
80%
76%
81%
74%
71%
79%
81%
77%
79%
76%
0.01
Whole blood
93%
96%
93%
93%
91%
93%
77%
94%
95%
92%
95%
93%
95%
93%
97%
89%
89%
85%
89%
90%
93%
91%
98%
93%
70%
90%
88%
97%
93%
93%
97%
95%
89%
90%
91%
90%
90%
92%
98%
0.1
106%
112%
108%
111%
126%
116%
83%
109%
110%
112%
103%
115%
111%
99%
111%
106%
122%
103%
106%
123%
123%
104%
115%
107%
81%
107%
217%
126%
109%
110%
102%
110%
107%
104%
108%
104%
103%
106%
103%
1
98%
103%
98%
99%
97%
96%
N.A
96%
99%
90%
105%
95%
100%
92%
92%
96%
86%
115%
95%
96%
79%
94%
105%
97%
77%
94%
86%
98%
101%
93%
99%
96%
94%
86%
94%
93%
94%
109%
99%
0.01
Urine
Alprazolam
Bromazepam
Brotizolam
Chlordiazepoxide
Clorazepic acid
Clotiazepam
Cloxazolam
Diazepam
Estazolam
Ethyl loazepate
Etizolam
Fludiazepam
Flunitrazepam
Flurazepam
Flutazolam
Flutoprazepam
Haloxazolam
Lorazepam
Lormetazepam
Medazepam
Mexazolam
Midazolam
Nimetazepam
Nitrazepam
Oxazolam
Prazepam
Quazepam
Rilmazafone
Tosopam
Triazolam
Zolpidem
α-Hydroxyalprazolam
α-Hydroxybrotizolam
α-Hydroxyetizolam (M-VI)
7-Aminounitrazepam
7-Aminonimetazepam
7-Aminonitrazepam
α-Hydroxytriazolam
Zolpidem M-1