100 / 658
6
Full Optimization of LC/MS Methods to Increase Robustness of Complicated Matrix Containing Samples Using Active Flow Management Chromatography
es) sample of 11-ketotestosterone
(Cmpd 3) and epitestosterone
column with PCS, a conventional
. column
Concentration
Alprazolam Alprazolam–
37
Cl
37
Cl/
35
Cl
TABLE 4. RSD% for alprazolam.
37
Cl species was not detected in all five replicate
samples and reported as not detected but was included in calibration curve.
Cmpd 3 Cmpd 4
2.76
7.01
5 73
11 79
(fg on column)
(peak area)
2
22.8
*
NA
6
14.7
15.9
0.266
60
3.4
9.2
0.261
.
.
1.75
3.48
.6 mm i.d. column by generating
on column in 30% crashed
200
0.9
3.2
0.251
600
1.2
1.3
0.248
2000
0.3
1.5
0.252
to remove solids). Samples were
ile phase A = 2 mM NH
4
OAc (aq),
e pump flow rate setting was
0 µL/min. Samples were detected
o transitions
m/z
309 → 281 and
c:\xcalibur\...\alp-neat-1_140525140355
05/25/1414:03:55
Blank
C:\Xcalibur\...\Alprazolam\ALP-Neat-10
05/25/1414:47:28
0.3 fg/uLAlprazolam
RT
011 108
SM
7G
FIGURE 7. TIC chromatograms of LOQ and LOD of alprazolam by curtain flow
chromatography. Also shown are the five replicate injections at the LOQ of 6 fg
on column showing excellent reproducibility in crashed plasma..
,
collision gas pressure of
weighting and is shown in
rally occurring
37
Cl parent ion
t linearity. %RSD indicates an
n. Figure 7 shows TIC
RT:
0.11 -1.08
SM:
7G
40
60
80
100
e Abundance
0
20
40
60
80
100
Relative Abundance
0.91
0.46
0.51
0.63
1.08
0.57
0.98
0.68
0.42
0.72
1.04
0.23
0.78
0.26
0.33 0.36
0.19
0.82
RT:0.50
AA:404
NL:2.25E1
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MS
alp-neat-
1_140525140355
NL:2.05E2
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MS Genesis
ALP-Neat-10
:
. - .
:
45
50
55
60
65
70
75
80
85
90
95
100
0.50
NL:2.12E2
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MS alp-neat-13
NL:1.77E2
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MS alp-neat-11
NL:1.57E2
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MS alp-neat-12
NL:1.60E2
TICF:+cESISRM
ms2309.000
[280999281001]
Blank
LOQ
6 fg on column
ducibility at the LOQ of 6 fg on
3. MS Acquisition conditions
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Time (min)
0
20
40
60
80
100
Relative Abundance
0
20
Relativ
RT:0.50
AA:169
NL:8.47E1
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MS Genesis
alp-neat-4
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Time (min)
0
5
10
15
20
25
30
35
40
.
- .
MS alp-neat-9
NL:2.05E2
TICF:+cESISRM
ms2309.000
[280.999-281.001]
MSALP-Neat-10
LOD
2 fg on column
e Conditions
tion
HESI +
e Voltage 600 V
emp
350 °C
To determine column robustness, 100 fg (in a 1:1 synthetic urine) was repeatedly
injected and peak areas were reported. Figure 8 shows peak shape and areas at
various stages of this analysis.
FIGURE 8. TIC chromatograms of various injections of 100 fg on column
mp
450 °C
HM 0.7
HM 0.7
alprazolam-stab-sweep-160
06/02/1417:32:30
10 fg/uLalprazolam insyntheticurine
RT:
0.20 - 0.70
0
20
40
60
80
100
RT: 0.42
AA: 960
RT: 041
NL: 4.40E2
TICF: + c ESISRM
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
10
NL: 393E2
RT:
0.20 - 0.70
0
20
40
60
80
100
RT: 0.42
AA: 904
RT: 042
NL: 4.13E2
TICF: + c ESISRM
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
160
NL: 424E2
alprazolam in 1:1 v/v synthetic urine, 20 µL injection
10 inj
160 inj
h d l
Time
0.3 min
Filter
3 s
0
20
40
60
80
100
0
20
40
60
80
100
RelativeAbundance
.
AA: 879
RT: 0.41
AA: 883
RT: 0.42
AA: 1006
.
TICF: + c ESISRM
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
50
NL: 4.08E2
TICF: + c ESISRM
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
100
NL: 4.56E2
TICF: +c ESISRM
0
20
40
60
80
100
0
20
40
60
80
100
RelativeAbundance
.
AA: 900
RT: 0.42
AA: 818
RT: 0.42
AA: 708
.
TICF: + c ESISRM
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
180
NL: 3.77E2
TICF: + c ESISRM
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
200
NL: 3.54E2
TICF: +c ESISRM
50 inj
100 inj
180 inj
200 inj
Conclusion
C t i fl
h t
h l
ff
i
ifi
t d t
i
iti it
s e p asma.
rally occurring)
: 1/X
0.2
0.3
0.4
0.5
0.6
0.7
Time (min)
0
20
40
60
80
100
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
150
0.2
0.3
0.4
0.5
0.6
0.7
Time (min)
0
20
40
60
80
100
ms2 309.000
[280.999-281.001] MS
Genesis
alprazolam-stab-sweep-
220
150 inj
220 inj
ur a n ow c roma ograp y co umns o ers s gn can a van ages n sens v y
and robustness over small-bore HPLC columns by eliminating wall effects that
lead to decreased column performance.
Curtain flow chromatography columns improve LC/MS research and forensic
methods especially where larger injection volumes are required and samples
contain complex matrices.
Future work requires reducing the actual inner diameter of the column to allow
virtual column inner diameters < 1mm, reducing overall flow rates and solvent
consumption. This can potentially show a large efficiency improvement where
micro-bore columns are more susceptible to wall effects than 2 1 mm i d
00
2000
: 1/X
.
. .
columns.
References
1 F Gritti G Guiochon
J Chromatogr A
1218 (2011) 1592
.
.
, .
,
.
.
.
2. R.A. Shalliker, M. Camenzuli, L. Pereira, H.J. Ritchie,
J. Chromatogr. A
,
1262 (2012) 64 AN64053.
0
2000
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to
encourage use of these products in any manners that might infringe the intellectual property rights of others.
PO64152-EN 0614S