glipizide (500 ng) glimepiride (500 ng) memory effects in...
TRANSCRIPT
min0 1 2 3 4 5
mAU
0
50
100
150
200
250
DAD (230 nm) Glipizide (500 ng)
Glimepiride (500 ng)
0 1 2 3 4 5 Tim e [min]0
1
2
3
4 x106 Intens.
Glipizide (25 ng) (m/z) 446 321 Da
Glimepiride (25 ng)(m/z) 491 352 Da
MS2 (APCI/ITD)
Memory effects in the interface
generate additional peak tailing!
Consequence:Enhance on
chromatographic resolution; more often increased resolution
means longer chromatographic runs !
Chromolith Performance RP-18e (100 x 4.6 mm), 25 oC; aq. 0.1% HCOOH / MeOH (30/70), 1 mL/min.
APCI/ Dry gas: Temp. 300 oC, 4 L/min; Vap. temp. 350 oC; Nebulizer gas press. 60 psi; Corona 4000 V; Vcap. 3500 V;
4x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
Abundance vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2
+ MRM (130.2 -> 60.2) Test04.d
1 1 2
4x10
00.25
0.50.75
11.25
1.51.75
22.25
2.52.75
33.25
3.53.75
44.25
4.54.75
Abundance vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8
+ MRM (130.2 -> 71.2) Test09.d
1 1 2
Target compound: Metformin – positive;
IS: 1-methylbiguanide– positive;
Column: Zorbax CN, 150 mm L x 4.6 mm i.d.; 5 µm d.p.; Agilent Technologies; Cat. No.:
883952-705; Cyanopropyl chemically modified, ultra-pure silica (>99.999%), end-
capped; S.N.: USJ0003915;
Column temperature: 25°C;
Mobile phase components:
Solvent A: Aqueous 10mM CH3COONH4 at pH= 3.5 with CH3COOH;
Solvent B: Acetonitrile;
Elution: isocratic;
Composition: Solvent A / Solvent B = 50 / 50;
Flow rate: 0.8 mL/min.
Injection volume: 5 µL (1 ng in column, each).
ESI IONIZATION PARAMETERS:
Drying gas temperature: 350°C;
Drying gas flow: 10 L/min;
Pressure of the nebulizer gas: 60 psi.;
Vcap: 1500 V.
Dwell: 50
Fragmentor: 100
Collision energy: 10
Delta EMV: 600
WORKING MODE: MS2 / MRM
Target compound: m/z = 130.2 to 60.2
IS: m/z = 116.2 to 60.2
Target compound: Metformin – positive;
IS: 1-methylbiguanide– positive;
Column: Zorbax Eclipse XDB-CN, 150 mm L x 4.6 mm i.d.; 3.5 µm d.p.; Agilent
Technologies; Cat. No.: 963967-905; Cyanopropyl chemically modified, ultra-pure
silica (>99.999%), end-capped; S.N.: USBAA01195;
Column temperature: 25°C;
Mobile phase components:
Solvent A: Aqueous 10mM CH3COONH4 at pH= 3.5 with CH3COOH;
Solvent B: Acetonitrile;
Elution: isocratic;
Composition: Solvent A / Solvent B = 20 / 80;
Flow rate: 0.8 mL/min.
Injection volume: 5 µL (1 ng in column, each).
ESI IONIZATION PARAMETERS:
Drying gas temperature: 350°C;
Drying gas flow: 10 L/min;
Pressure of the nebulizer gas: 60 psi.;
Vcap: 1500 V.
Dwell: 50
Fragmentor: 100
Collision energy: 10
Delta EMV: 600
WORKING MODE: MS2 / MRM
Target compound: m/z = 130.2 to 60.2
IS: m/z = 116.2 to 60.2
IONIZATION YELDS
Depending upon M.Ph. composition!
Poor reproducibility with respect to other spectrometric detectors!
0 100 200 300 400 500 600 700350
400
450
500
550
600
650
700
750
800
850
900
950
1000
Average
Average - 2*s (for RCV=2.46%)
Average + 2*s (for RCV=2.46%)
Average - 2*s (for RCV=15%)
Average + 2*s (for RCV=15%)
IS p
eak
area
(L.U
.)
Sample number
rxy = -0.2
0 100 200 300 400 500 6008
10
12
14
16
18
20
22
IS p
eak
area
(mAu
)Sample number
Average + 2*s (for RCV=15%)
Average + 2*s (for RCV=6.64%)
Average - 2*s (for RCV=6.64%)
Average - 2*s (for RCV=15%)
Average
rxy= 0.2
Trends for I.S. peak area values
Gliquidone BE study I.S. – Fluoranthene; 644 samples real plasma samples Zorbax SB-C18 50 mm x 4.6 mm x 1.8 m; 60 oC Vinj = 50 L; Aq. 0.1% H3PO4 / ACN (70/30); gradient elution to 100% ACN; Flow rate: 2 mL/min; Ex. 230 nm; Em. 415 nm; duration – 1.8 min.
Tenoxicam BE study I.S. – Piroxicam; 560 samples real plasma samples Zorbax SB-C18 50 mm x 4.6 mm x 1.8 m; 60 oC Vinj = 50 L; Aq. 0.1% H3PO4 / ACN (70/30); gradient elution to 100% ACN; Flow rate: 2 mL/min; UV - 368 nm (resp. time 0.1 s); duration – 4 min.
0 100 200 300 400 500 600 70040000
50000
60000
70000
80000
90000
100000
110000
120000
130000
Average - 2*s (for RSD=15%)
Average + 2*s (for RSD=15%)
Average - 2*s (for RSD=5.63%)
Average + 2*s (for RSD=5.63%)
IS p
eak
area
Sample number
Averagerxy = 0.81
Trends for I.S. peak area values
0 200 400 600 80060000
80000
100000
120000
140000
160000
180000
200000
Average
Average - 2*s (for RSD=11%)
Average + 2*s (for RSD=11%)
Average - 2*s (for RSD=15%)
Average + 2*s (for RSD=15%)
IS p
eak
area
Sample number
rxy = -0.8
Bromazepam BE study I.S. – Nitrazepam; 644 samples real plasma samples Zorbax SB-C18 Rapid Resolution 30 mm x 2.1 mm x 3.5 m; 25 oCVinj = 5 L Aq. 0.1% HCOOH / MeOH (60/40); isocratic; Flow rate: 0.8 mL/min(+)AP-ESI/MS2 (QQQ): Nitrazepam (m/z) 282316 Da; Bromazepam (m/z) 282316 Da Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 4000 V.
Metformin BE study I.S. – 1-methylbiguanide; 832 samples real plasma samples Zorbax CN 150 mm x 4.6 mm x 5 m; 25 oC Vinj = 5 L; Aq. 0.1% 10 mM acetate buffer pH=3.5 / ACN (50/50); isocratic; Flow rate: 0.8 mL/min (+)AP-ESI/MS2 (QQQ): I.S. (m/z) 11660 Da; Metformin (m/z) 13060 Da Dry gas temp.: 325 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 1500 V.
0 200 400 600 800 1000160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
Average
Average - 2*s (for RSD=13.2%)
Average + 2*s (for RSD=13.2%)
Average - 2*s (for RSD=15%)
Average + 2*s (for RSD=15%)
IS p
eak
area
Sample number
rxy= -0.92
source cleaning
Trends for I.S. peak area values
Meloxicam BE studyI.S. – 4-hydroxy-2-methyl-N-(pyridine-2-yl)-2-H-1,2-benzothiazine-3-carboxamide-1,1-dioxide ; 936 samples real plasma samples Zorbax Eclipse XDB-C18 15 mm x 4.6 mm x 5.5 m; 25 oC Vinj = 5 L; Aq. 0.2% HCOOH / ACN (30/70); isocratic; Flow rate: 0.8 mL/min (+)AP-ESI/MS2 (QQQ): I.S. (m/z) 33295 Da; Meloxicam (m/z)352115 Da Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 3000 V.
0 100 200 300 400 500 600 7001000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
11000000
Average
Average - 2*s (for a RSD=36,3%)
Average + 2*s (for a RSD=36,3%)
Average - 2*s (for a RSD=15%)
I.S. p
eak
area
Sample number
Average + 2*s (for a RSD=15%)
cleaning source
Glibenclamide BE studyI.S. – gliquidone ; 624 samples real plasma samples Chromolith Performance RP-18e, 10 mm x 4.6 mm; 40 oC Vinj = 50 L; Aq. 0.1% HCOOH / ACN (42/58); isocratic; Flow rate: 1.0 mL/min (+)APCI/MS2 (ITD): I.S. (m/z) 528403 Da; Glibenclamide (m/z) 494369 Da Dry gas temp.: 300 oC; flow 5 L/min; Nebulizer gas press. 60 psi, Corona 5000 V; V cap. 4500 V.
Consequence: response stability will directly influence on the frequency of running calibrations and QC samples sets!
LUOL Calibration
y = 0.1226x + 0.029R2 = 0.9999
0.0000
2.0000
4.0000
6.0000
8.0000
10.0000
12.0000
14.0000
0 20 40 60 80 100 120
Concentration (ppb)
Ana
lyte
/I.S.
pea
k ar
ea ra
tio
Concentration (ppb) Analyte/I.S. peak ratio RSD%(n=6) Backinterpolated
Concentration (ppb) %Bias
0 0.0000 - - -0.5 0.0814 2.76 0.43 -14.6
1 0.1426 4.59 0.9 -7.42.5 0.3231 0.32 2.4 -4.1
5 0.6053 0.61 4.7 -6.010 1.2646 1.26 10.1 0.825 3.2175 3.22 26.0 4.050 6.1437 6.14 49.9 -0.3
100 12.2664 12.27 99.8 -0.2
N
NHOH
H
O
H
CH3
CH3
Non-linear response functions!
Consequence:Find ways of
making linearisation !
Nicergoline BE study
I.S.–1,3’-(carboxypropyl)-3,7-dimethyl xanthine;
Also analyzed: metabolites LUOL and MELUOL
Zorbax Eclipse XDB-C8, 150 mm x 4.6 mm x 3.5 m; 40 oCVinj = 300 L; Aq. 0.1% HCOOH / 0.1% HCOOH in MeOH (74/26); gradient elution; Flow rate: 0.8 mL/min
(+)APCI/MS2 (ITD): LUOL (m/z) 287255+269 DaDry gas temp.: 250 oC; flow 5 L/min; Vaporizer temp. 350 oC; Nebulizer gas press. 60 psi, Corona 5000 V; V cap. 4500 V.
12 14
0.0
1.0
1.5
2.0x105
0 2 4 6 8 10 [min]
0.5
Intens.
DAcDLTZ
IS
DMeDLTZ
DLTZ
N
S
NH+
H
O
OO
O
NH+
SH
O
OO
O
NH+
S
O
OO
S
C+
O
N
O
S+
O
N
S+
C
N
OH
O
S
O
N
CH
+
NSH
+
O
C+
O
SH
N
C+
SHN
C6H4 OMe
- HN(CH3)2
m/z=415 m/z=370
- CH3COOH
m/z=310
m/z=178
- CO
m/z=150
-
Diltiazem BE study
I.S. – diastereoisomer of DLTZ; Also analyzed: metabolites Desacetyldiltiazem (DAcDLTZ) and Desmethyldiltiazem (DMeDLTZ) Zorbax Eclipse XDB-C8, 150 mm x 4.6 mm x 5 m; 45 oC Vinj = 500 L; Aq. 0.2% TEA at pH=3.5 with HCOOH / ACN (75/25); isocratic; Flow rate: 0.8 mL/min (+)ESI/MS2 (ITD): IS = DLTZ: (m/z) 415370, 310, 178, 150 Da (isolation window 408 ± 10 Da) DAcDLTZ: (m/z) 373328 + 223 + 178 + 150 Da (isolation window 373 ± 2 Da) DMeDLTZ: (m/z) 401370, 341, 310, 178, 150 Da (isolation window 408 ± 10 Da) Dry gas temp.: 365 oC; flow 12 L/min; Nebulizer gas press. 65 psi.; V cap. 1500 V.
Limited resolution (IS, DMeDLTZ, DLTZ) single isolation window broad isolation one (± 10 Da) no CID fragmentation Product ions Precursor
ions !
DLTZ
y = 0.0073x + 0.1059R2 = 0.9822
y = -9E-06x2 + 0.0105x + 0.0341R2 = 0.9985
0
0.5
1
1.5
2
2.5
3
3.5
0 50 100 150 200 250 300 350 400 450
Concentration (ppb)
Ana
lyte
/I.S
. pea
k ar
ea ra
tio
Con
cent
ratio
n (p
pb)
Ana
lyte
/I.S.
pea
k ar
ea
ratio
Bac
kint
erpo
late
d co
ncen
trat
ion
valu
es
(LIN
EAR
)
% B
ias
Bac
kint
erpo
late
d co
ncen
trat
ion
valu
es
(BIN
OM
IAL)
% B
ias
0.5 0.01813 -12.1 -2519.3 -1.5 -403.8 1 0.02464 -11.2 -1219.8 -0.9 -190.0 5 0.08249 -3.2 -164.4 4.6 -7.5
10 0.16973 8.8 -11.9 13.1 30.6 25 0.30335 27.2 8.9 26.2 4.9 50 0.56244 63.0 25.9 52.7 5.4
100 1.05993 131.6 31.6 107.6 7.6 200 1.72370 223.1 11.5 192.8 -3.6 400 2.85453 379.0 -5.3 419.3 4.8
Non – linear calibration !Linear versus Binomial
DLTZ
y = 0.7848x - 1.5799R2 = 0.9982
-2.000
-1.500
-1.000
-0.500
0.000
0.500
1.000
-0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00
Log(concentration)
Log(
anal
yte/
I.S. p
eak
area
ratio
)
Con
cent
ratio
n (p
pb)
Log
(con
cent
ratio
n)
Ana
lyte
/I.S.
pea
k ar
ea ra
tio
Log
(Ana
lyte
/I.S.
pe
ak a
rea
ratio
)
Bac
k in
terp
olat
ed
conc
entr
atio
n va
lues
% B
ias
0.5 -0.30 0.017 -1.766 0.6 12.8 1 0.00 0.025 -1.608 0.9 -10.1 5 0.70 0.083 -1.078 4.3 -14.2
10 1.00 0.170 -0.770 10.7 6.5 25 1.40 0.303 -0.518 22.4 -10.3 50 1.70 0.562 -0.250 49.5 -1.1
100 2.00 1.060 0.025 111.4 11.4 200 2.30 1.724 0.236 207.7 3.9 400 2.60 2.855 0.456 396.5 -0.9
Non – linear calibration !Log – Log representation
Non – linear calibration ! Log – Log representationCarbamazepina - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 6 QCs
Concentration (ppb)
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
y = -1.9875E-008 * x ^ 2 + 8.1426E-004 * x + 0.0202R^2 = 0.99973470
Calibration
y = 0,91256x - 2,82874R2 = 0,99938
-1,500
-1,000
-0,500
0,000
0,500
1,000
1,500 2,000 2,500 3,000 3,500 4,000 4,500
log Carbamazepine concentration (ng/mL)
log
Peak
are
a ra
tio C
arba
maz
epin
e / I
.S.
Internal Standard: 7-nitro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one– positive;
Target compound 1: Carbamazepine- 5H-Dibenzo[b,f]azepine-5-carboxamide– positive;
Target compound 2: Carbamazepine 10,11-epoxide- positive;
Column: Zorbax Eclipse XDB-C18, Rapid Resolution, 50 mm L x 4.6 mm i.d.; 1.8 µm
d.p.; Octadecyl chemically modified, ultra-pure silica (>99.995%), double end-
capped; S.N.:USWDY04740; Agilent Technologies; Cat. No.:927975-902;
Column temperature : 25°C;
Solvent A: 0.1% HCOOH;
Solvent B: Acetonitril;
Elution: isocratic; Composition of the mobile phase: Solvent A / Solvent B = 70/ 30;
Flow rate : 0.8 mL/min.
Injection volume : 5 µL.
(+) ESI
Drying gas temperature: 350°C;
Drying gas flow: 12 L/min;
Pressure of the nebulizer gas : 55 psi;
Vcap: 1500 V.
Collision Energy: 25 V.
Fragmentor: 90 V.
ΔEMV: 600 V.
MRM
IS: m/z = 282.0 to 236.0 (qual. 254.0);
Target compound 1: m/z = 237.0 to 194.0 (qual. 192.0);
Target compound 2: m/z = 253.0 to 180.0 (qual. 210.0).
Concentrations (ng/mL):
I.S.: 1000 ;
Target compound 1: Concentrations (ng/mL):1) 100; 2) 200; 3) 500; 4) 1000; 5) 2000; 6)
3500; 7) 5000; 8) 10000.
Non – linear calibration (Log – Log representation) – Results!
Real samples for 27 volunteers
0
0.0002
0.0004
0.0006
0.0008
0.001
0.0012
0 200 400 600 800 1000 1200 1400
Sample number
Res
pons
e to
con
cent
ratio
n ra
tio
(nor
mal
ized
ratio
)
P1
P2
MSD
waste
C1
C2
Inj NH2
CH3
OH
Cathine [(1S,2S)-2-amino-1-phenylpropane-1-ol] CAS 492-39-7
Study of Ephedrines in Urine Target analyte: Cathine; I.S.: 4-aminoantipyrine Also analyzed: norephedrine, ephedrine, pseudoephedrine, N-methylephedrine C1 – Oasis HLB (polyvinylpyrrolidone) 20 mm x 3 mm x 5 m; 25 oC; Vinj = 100 L; Direct injection of filtered urine. C2 – Purosphere Star C18, 125 mm x 4 mm x 5 m; 25 oC M. Ph. 1: Aq. 0.015% HCOOH at pH 10.5 with TEA; isocratic elution; M. Ph. 2: Aq. 0.015% HCOOH at pH 10.5 with TEA / MeOH (55/45); isocratic; Flow rate: 1.0 mL/min (+)ESI/MS2 (QQQ): Cathine (m/z) 134117; 91 Da; NO MOLECULAR ION IN THE ESI SOURCE! Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 3500 V.
OH2+
CH3
NH2CH
+
CH3
NH2
CH
C+
CH3
CH
CH3
NH3+
C+
CH2
CH2+152134
117
- NH3- H2
115
- C2H2
91
Non – linear calibrations ! Analysis of Ephedrines in Urine(+) ionisation despite alkaline mobile phase character; Fragmentation within the source.
CATHINE
y = 0.0139x2 + 0.0059x + 0.0044R2 = 0.9992
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 10 12Concentrations (ppm)
Ana
lyte
/I.S.
pea
k ar
ea ra
tio
Con
cent
ratio
n (p
pm)
Ana
lyte
/I.S.
pea
k ar
ea ra
tio
Bac
k in
terp
olat
ed
conc
entr
atio
n va
lues
% B
ias
0 0 - - 0.625 0.0158 0.7 14.9 1.25 0.0398 1.4 11.8
2.5 0.1104 2.6 2.3 5 0.3568 4.8 -3.5
7.5 0.8558 7.6 1.6 10 1.4465 10.0 -0.2
Non – linear calibration !Binomial representation
n
xB
n
yA
n
xx
n
yxyx
B
n
ii
n
ii
n
i
n
ii
i
n
i
n
ii
n
ii
ii
11
1
2
12
1
11
LINEAR
Con
cent
ratio
n (p
pb)
Ana
lyte
/I.S.
pea
k ar
ea
ratio
Bac
k in
terp
olat
ed
conc
entr
atio
n va
lues
% B
ias
25 0.0574 -42.4 -269.7 50 0.1105 -9.2 -118.5
100 0.2088 52.2 -47.8 500 1.0657 587.7 17.5
1000 1.9468 1138.4 13.8 1500 2.7224 1623.2 8.2 2000 3.3371 2007.3 0.4 2500 4.0217 2435.3 -2.6
METFORMIN
y = 0.0016x + 0.1253R2 = 0.992
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 500 1000 1500 2000 2500 3000
Concentration (ppb)
Ana
lyte
/I.S.
pea
k ar
ea r
atio
Metformin BE study
I.S. – 1-methylbiguanide; Zorbax CN 150 mm x 4.6 mm x 5 m; 25 oC Vinj = 5 L; Aq. 0.1% 10 mM acetate buffer pH=3.5 / ACN (50/50); isocratic; Flow rate: 0.8 mL/min
(+)AP-ESI/MS2 (QQQ): I.S. (m/z) 11660 Da; Metformin (m/z) 13060 Da Dry gas temp.: 325 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 1500 V.
Non-linearCalibrations
Weighing by 1/x or 1/x2!
Increasing linearity domain!
ii
n
ii
n
iii
n
ii
n
iii
n
in
ii
n
iii
ii
n
in
ii
n
iii
n
iii
iii
xw
w
wxB
w
wyA
w
wxwx
w
wywxwyx
B
1
1
1
1
1
1
1
2
12
1
1
11
LINEAR - Weighing 1/x Concentration (ppb) Analyte/I.S. peak area ratio Back interpolated concentration values % Bias
25 0.0574 18.2 -27.450 0.1105 48.9 -2.2
100 0.2088 105.8 5.8500 1.0657 601.6 20.3
1000 1.9468 1111.5 11.21500 2.7224 1560.4 4.02000 3.3371 1916.1 -4.22500 4.0217 2312.3 -7.5
Metformin weighted 1/x
y = 0.001728x + 0.026043R^2 = 0.9923
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 500 1000 1500 2000 2500 3000Concentration (ppb)
Ana
lyte
/I.S.
pea
k ar
ea ra
tio
ii
n
ii
n
iii
n
ii
n
iii
n
in
ii
n
iii
ii
n
in
ii
n
iii
n
iii
iii
xw
w
wxB
w
wyA
w
wxwx
w
wywxwyx
B
1
1
1
1
1
1
1
2
12
1
1
11
Metformin weighted 1/x2
y = 0.001846x + 0.013402R^2 = 0.9893
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 500 1000 1500 2000 2500 3000Concentration (ppb)
Ana
lyte
/I.S.
pea
k ar
ea ra
tio
LINEAR - Weighing 1/x2 Concentration (ppb) Analyte/I.S. peak area ratio Back interpolated concentration values % Bias
25 0.0574 23.8 -4.6 50 0.1105 52.6 5.2
100 0.2088 105.9 5.9 500 1.0657 570.0 14.0
1000 1.9468 1047.3 4.7 1500 2.7224 1467.5 -2.2 2000 3.3371 1800.5 -10.0 2500 4.0217 2171.4 -13.1
Amlodipine BE study
I.S. – (3-Ethyl-5-methyl-(4-R,S)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl- 1.4-dihydropyridine-3,5-dicarboxilate); Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm x 3.5 m; 40 oC Vinj = 200 L; Aq. 0.1% HCOOH / ACN (60/40); isocratic elution; Flow rate: 0.8 mL/min (+)ESI/MS2 (QQQ): Amlodipine (m/z) 409 238 Da; I.S. (m/z) 321 167 Da Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi; V cap. 4000 V.
x102
0
1
2
3
4
Abundance vs. Acquisition Time (min)
0.5 1 1.5 2 2.5 3 3.5
+ EIC MRM (** -> 238.0) test419.d 3.2301 2 2
LLOQ sample
Residual peak in blank samples
%20100samples LLOQin AreaPeak
samplesBlank in areaPeak Residual LLOQ Confirm.
Sometimes, too high sensitivity
is obtained !
Consequence:Residual peak areas in blank
samples limitatemethod LLOQ.
20 ppb amlodipine in water
20 ppb amlodipine spiked to deproteinized blank plasma
Yield ~ 30 %
20 ppb amlodipine spiked to plasma and then deproteinized
Yield ~ 30 %
x104
0
1
2
3
4
5
Abundance vs. Acquisition Time (min)
0.5 1 1.5 2 2.5 3 3.5
+ EIC MRM (** -> 238.0) 2.8921 1 2 2
Sam
ple
20 n
g/m
L (w
ater
)
20 n
g/m
L (d
epro
tein
ized
bla
nk
plas
ma)
20 n
g/m
L (b
lank
pl
asm
a)
Peak Areas 1 202,864 72,348 84,040 2 235,531 74,147 80,329 3 261,237 76,714 77,511 4 294,921 83,399 81,939 5 285,747 84,433 82,091 6 333,760 85,658 83,603
Average 274,010 79,450 81,586 s 49,910 5,745 2,394
RSD % 14.21 7.23 2.93 Recovery within samples in water and plasma matrix (%) 29.8
Recovery within samples with plasma matrix (%) 102.7
Signal suppression due to co-extracted plasma matrix !Consequences: Capacity factors for target compounds higher than 5 are strongly recommended.
Sometimes, signal enhancement may be observed. In such cases, “addition method” may be considered as a practical solution.
# Type of
anticoagulation agent in plasma
sample Am
lodi
pine
co
nc. (
ng/m
L)
Analyte / I.S. peak area ratio
Cal
cula
ted
conc
.
Mea
n co
nc.
RSD
%
% B
ias
1. 1.0387 8.7 2. 1.1093 9.3 3. 1.1182 9.3 4. 1.0382 8.7 5. 1.2087 10.1 6.
Li-Heparin 7.5
1.0814 9.0
9.2 5.8 22.4
7. 0.9523 8.0 8. 0.9357 7.8 9. 0.9856 8.2 10. 1.0142 8.5 11. 1.0272 8.6 12.
K-EDTA 7.5
1.0234 8.6
8.3 3.9 10.2
13. 0.8297 6.9 14. 0.8400 7.0 15. 0.8245 6.9 16. 0.8456 7.1 17. 0.8655 7.2 18.
Citrate 7.5
0.8663 7.2
7.1 2.1 - 5.8
Influence of the anticoagulation agent should be
carefully studied !
Enhancement / supressioneffects may
arrise!
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
500
1000
1500
2000
2500 MWD1 B, Sig=238,4 Ref=480,20
min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
-75
-50
-25
0
25
50
75
100 MWD1 B, Sig=238,4 Ref=480,20
UV trace on C1
UV trace on C2
P1
P2
MSD
waste
C1
C2
Inj
Felodipine BE Study Target analyte: Felodipine; I.S.: Impurity C of Felodipine C1 – LiChrosphere ADS RP-18 (shielded stationary phase) 30 mm x 4.6 mm x 5 m; 35 oC; Vinj = 500 L; Direct injection of filtered plasma.C2 – Zorbax Eclipse XDB C18, 150 mm x 4.6 mm x 3.5 m; M. Ph. 1: Aq. 0.1% H3PO4 at pH 5 with TEA / MeOH (98/2); Flow rate: 0.8 mL/min; gradient elution (composition + flow rate); M. Ph. 2: Aq. 0.1% HCOOH / MeOH (27.5/72.5); Isocratic elution; Flow rate: 1.0 mL/min (+)APCI/MS2 (ITD): Felodipine (m/z) 384 356 + 354 + 352 + 340 + 338 Da; I.S. (m/z) 398 370 + 368 + 352 + 342 + 340 Da; Dry gas temp.: 300 oC; flow 5 L/min; Vaporizer temperature 350 oC; Nebulizer gas press. 60 psi.; Corona 6000 V; V cap. 3500 V.
Less SamplePreparation
means a serious
improvementof the
process overall
reprodu-cibility!
Volunteer
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 10 20 30 40 50
Time (hours)
Felo
dipi
ne C
once
ntra
tion
(ng/
mL)
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time [min]
0.0
0.5
1.0
1.5
x106 Intens.
I.S.
Felodipine
Blank Plasma Sample
Spiked Plasma Sample (10 ng/mL)
High sensitivity (tenths of ppt) and required reproducibility is reached with a less performant mass analyzer (such as IT)!
Consequences: Costs are traduced in longer separation times and complicated experimental set-up.
1x10
0
1
2
3
4
Abundance vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
+ MRM (609.3 -> 195.1) ReserpineCheckout001.d Smooth (1) Noise (ASTM) = 2.17; SNR (1.497min) = 22.6
1.4971 1
1x10
0
1
2
3
4
5
+ MRM (609.3 -> 195.1) ReserpineCheckout002.d Smooth (1) Noise (ASTM) = 0.58; SNR (1.507min) = 99.3
1.5071 1
1x10
00.5
11.5
22.5
33.5
4
+ MRM (609.3 -> 195.1) ReserpineCheckout003.d Smooth (1) Noise (ASTM) = 0.81; SNR (1.497min) = 52.0
1.4971 1
1x10
00.5
11.5
22.5
33.5
4
+ MRM (609.3 -> 195.1) ReserpineCheckout004.d Smooth (1) Noise (ASTM) = 0.97; SNR (1.517min) = 42.3
1.5171 1
1x10
00.5
1
1.52
2.53
3.5
+ MRM (609.3 -> 195.1) ReserpineCheckout005.d Smooth (1) Noise (ASTM) = 1.67; SNR (1.507min) = 24.0
1.5071 1
1 2
3
4
5
6
0 1 2 3 4 5 6 7 8
Time [min]
0
1
2
3
4
5
x104Intens. 5 pg of reserpine in the interface
5 fg of reserpine in the interface
RSD% = 14.3% RSD% = 7.1%RSD% <= 15%
QQQ IT
The inherent sensitivity and reproducibility of the mass analyzer !
180
160
140
120
100
80
60
40
20200 180 160 140 120 100 80 60 40 20
Conc. (ng/mL) on ESI / ITD
Con
c. (n
g/m
L) o
n A
PCI /
ITD
Conc. (ng/mL) on ESI / QQQ (1)
Conc. (ng/m
L) on ESI / QQ
Q (2)
rxy = 0.8279
rxy = 0.8279 rxy = 0.8731
rxy = 0.9958
20
40
60
80
100
120
140
160
180
200
20 40 60 80 100 120 140 160 180 200
BROMAZEPAM BE Study Analyte: Bromazepam; I.S. Nitrazepam
Sample preparation: Protein precipitation with ACN (Plasma / ACN = 1 / 2) Samples from 1 volunteer (all range of concentrations); 4 aliquots for each sample; 2 aliquots by ESI/QQQ; 1 aliquot by
ESI/ITD; 1 aliquot by APCI/ITD; for each series of data one calibration and QC sets (n=6) have been considered. Zorbax SB-C18; 30 mm x 2.1 mm x 3.5 mm; 25 oC Purospher Star C-18; 125 mm x 4 mm x 5 mm; 25 oC Vinj = 5 mL Vinj = 200 mL Aq. 0.1% HCOOH / MeOH (60/40), Flow 0.8 mL/min (k’ = 7.2 for Bromazepam, 11.2 for I.S.).
Aq. 0.1% HCOOH / MeOH (56/44), Flow 0.8 mL/min, in order to obtain same k’
(+) ESI/MS2 (QQQ); (+) ESI/MS2 (ITD); (+) APCI/MS2 (ITD) Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 4000 V.
Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 2535 V. Dry gas temp.: 350 oC; flow 10 L/min; Vaporizer temperature: 350 oC Nebulizer gas press. 60 psi.; Corona 4000 V; V cap. 3000 V.
Analyte: m/z (316/318) 182 Da; I.S.: m/z (282) 180 Da;
Different ion sources!
Different mass analyzers!
Apparently, IP separation mechanism is useless with MSD! In practice, perfluorurated IPA may be used!
Analysis of Ephedrines in Urine Samples Target analytes: Cathine; Norephedrine; Ephedrine; Pseudoephedrine; N-Methylephedrine;I.S.: 4-aminoantipyrine
Luna C18(2) 250 mm x 4.6 mm x 5 m; 25 oC; Vinj = 5 L; Non-extractive sample preparation technique.M. Ph.: Aq. 0.15% HEPTAFLUOROBUTYRIC ACID (HFBA) / MeOH / I-PrOH (70/24/6); Flow rate: 0.8 mL/min; isocratic elution.
(+)ESI/MS2 (QQQ): Cathine + Norephedrine (m/z) 134 91 (cant.); 115 (cal.) Da;Ephedrine + Pseudoephedrine (m/z) 166 148 (cant.); 117 (cal.) Da;N-Methylephedrine (m/z) 180 162 (cant.); 147 (cal.) Da;I.S. (m/z) 204 56 (cant.); 94 (cal.) Da;
Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 3500 V.
I.S.
Cathine
Norephedrine
Ephedrine
Pseudoephedrine Methylephedrine
TRENDS FOR CATHINE PEAK AREA (HFBA)
y = -381.2x + 66055R2 = 0.9203
45000
50000
55000
60000
65000
70000
75000
0 10 20 30 40 50 60
Sample No.
Cat
hine
Pea
k A
rea
TRENDS FOR CATHINE PEAK AREA (HCOOH)
y = -15.975x + 12403R2 = 0.1395
0
5000
10000
15000
20000
25000
0 5 10 15 20 25 30 35 40
Sample numberC
athi
ne P
eak
Are
a
But….
Problems always may be solved … “chromatographically” !
I.S.Cathine
Pseudoephedrine
Ephedrine
Methylephedrine
Norephedrine
Analysis of Ephedrines in Urine Samples Target analytes: Cathine; Norephedrine; Ephedrine; Pseudoephedrine; N-Methylephedrine;I.S.: 4-aminoantipyrine
Betasil Phenyl 250 mm x 4.6 mm x 5 m; 25 oC; Vinj = 5 L; Non-extractive sample preparation technique.M. Ph.: Aq. 0.1% HCOOH / MeOH (78/22); Flow rate: 0.8 mL/min; isocratic elution.
(+)ESI/MS2 (QQQ): Cathine + Norephedrine (m/z) 134 91 (cant.); 115 (cal.) Da;Ephedrine + Pseudoephedrine(m/z) 166 148 (cant.); 117 (cal.) Da;N-Methylephedrine (m/z) 180 162 (cant.); 147 (cal.) Da;I.S. (m/z) 204 56 (cant.); 94 (cal.) Da;
Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 3500 V.
0 8
x10
4 10 12 Time [min]
0.00
0.25
0.50
1.00
Intens.
6
0.75
1.25
8
2
(+) APCI / MS2(+) APCI / MS (EIC)
(+) ESI / MS (EIC)
(+) ESI / MS2
(-) ESI / MS2
Lova
stat
ic A
cid
Sim
vast
atic
Aci
d
Lova
stat
in
Sim
vast
atin
Analyte Decomposition in the Ion Source !
Agreement between the ionization pattern and the type of the mass analyzer!
60.5
60 80 100 120 140 160 180 m/z0.0
0.5
1.0
1.5
6 x10
Intens
NH3+
NH
NCH3
CH3
NH NH
MS
60.4
85.1
113.1
130.1
60 80 100 120 140 160 180 m/z0.0
0.5
1.0
1.5
2.0
2.5
3.0 4 x10
Intens.
NH2 NH2
NH2+
130.1
(MS)2
[M+H+]
Metformin BE Study in Metformin / Glibenclamide combinations (only ITD available to date)Target analytes: Metformin; Glibenclamide; I.S.: Metformin Impurity D; Gliquidone Zorbax CN, 150 mm x 4.6 mm x 5 m; 25 oC; Vinj = 50 L; Sample preparation based on protein precipitation. M. Ph.: Aq. 10 mM NH4OOCCH3 at pH 3.5 with CH3COOH / ACN (50/50); Flow rate: 1.0 mL/min; Isocratic elution; (+)APCI/MS2 (ITD): Metformin TIC (m/z) 130 Da (EIC); I.S. TIC (m/z) 127 Da (EIC); Dry gas temp.: 300 oC; flow 5 L/min; Vaporizer temperature 350 oC; Nebulizer gas press. 60 psi.; Corona 4000 V; V cap. 2336 V.
0 1 2 30.0
0.2
0.4
0.6
0.8
1.0
7x10
Intens
Time [min] 4 5
I.S. 1
Met
form
in
I.S. 2
Glib
encl
amid
e
(+) MS
(+) MS2
50 ng
SIMVASTATIN
Precursor ion: m/z = 441.0; Product ions: m/z = 325.0
LOVASTATIN
Precursor ion: m/z = 405.0; Product ions: m/z = 199.0; 225.0; 243.0; 267.0; 285.0; 303.0.
282.3
419.1441.2
0.0
0.5
1.0
1.5
2.0
2.5 x106 Intens.
200 250 300 350 400 450 m/z
325.2
441.2
0.0
0.5
1.0
1.5
x106Intens.
200 250 300 350 400 450 m/z
199.1 267.1
285.1
303.1 387.1
405.1
200 225 250 275 300 325 350 375 400 425 m/z0
1
2
3
4
5 x105
Intens. 199.1
225.1243.1
267.1
285.1
303.2
200 225 250 275 300 325 350 375 400 425 m/z0.0
0.2
0.4
0.6
0.8
1.0 x105Intens.
O
H CH3
O
H
O
CH3CH3 CH3
H
O
H
OHHHCH3 H
O
H CH3
OH
O
CH3CH3
H
O
H
OHHHCH3 H
H
Cluster formation !
System dependent
phenomena!
In MS2,the choice of the right precursor
ion is essential for
the reproduci-
bility of quantitative
data.
Simvastatin BE Study
Target analytes: Simvastatin + Simvastatic Acid; I.S. 1: Lovastatin; ; I.S. 2: Lovastatic Acid
Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm x 3.5 m; 25 oC; Vinj = 50 L; M. Ph.: Aq. 0.1% HCOOH / ACN (35/65); Flow rate: 0.8 mL/min; Isocratic elution;
ESI/MS2 (ITD): Simvastatin (m/z) 441 325 Da (+);I.S. 1 (m/z) 405 199 + 225 + 243 + 267 + 285 + 303 Da (+);Simvastatic Acid
(m/z) 435 319 Da (-);I.S. 2
(m/z) 421 319 Da (-);
Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 70 psi.; V cap. 3500 V.
Reproducibility = f(precursor ion)
0
10000000
20000000
30000000
40000000
50000000
60000000
70000000
80000000
0 5 10 15 20 25 30
Sample no.
Peak
Are
a
Molecular ions are precursorsNa - adducts ions are precursors
Lovastatin RSD%=16.6%
Lovastatin RSD% = 4.32%
Simvastatin RSD% = 33.1%
Simvastatin RSD%=4.7%
Signal reproducibility is better for
Simvastatin when the Na adduct ion is
chosen as precursor ion while for Lovastatin
the molecular [M+H]+
precursor ion provides better results.
And all this long story, only for … a methyl
group!
Conclusions:
1. In LC/MS no advantage is taken for free. Everytime you had to pay for it!
2. LC/MS methods should be attentively validated before a large scale application. During a large scale application, periodic checkings for linearity and accuracy are mandatory (don’t forget to justify frequency)!
3. MSD imposes simple and direct sample preparation methods. Otherwise, closely follows on the reproducibility of results.
4. Is better to focus on understating phenomena. In depth systemoperating abilities are necessary, but this comes after!