Capillary electrophoresis
focusing on chiral separation
Mass spectrometry
MS, GC-MS, LC-MS
Lukáš Plaček
2 Proprietary & Confidential
Osnova prezentace
1. General terms in analytical chemistry
(10´; 1 question)
2. Capillary electrophoresis
(35´; 2 questions)
3. Mass spectrometry
• In general (25´; 2 questions)
• GC-MS (10´; 1 question)
• HPLC-MS (10´; 1 question)
3 Proprietary & Confidential
3
4 Proprietary & Confidential
Min
0 0,30
1
2 3
4
5
0,05 0,10 0,15 0,20 0,25
30.0 %
60.0
30.0
0,35 0,40
5 Proprietary & Confidential
6 Proprietary & Confidential
GENERAL TERMS
IN ANALYTICAL CHEMISTRY
7 Proprietary & Confidential
1. General terms – Qualitative vs. Quantitative analysis
2. Calibration curve, sensitivity of method
3. Noise, limit of detection, limit of quantitation
4. Accuracy/precision, repeatibility/reproducibility
8 Proprietary & Confidential
Terms
QUALITATIVE
(what?)
QUANTITATIVE
(how much/many?)
• identification of unknown sample
• identification of flammables
• identification of umpurities in Paralen
• determination of Cd in milk
• determination of pervintine in blood
• determination of Hg in hair
Sample, analyte
9 Proprietary & Confidential
Sensitivity
Concentration of analyte
Signal
Sensitivity = slope of calibration curve
Calibration curve
10 Proprietary & Confidential
LOD, LOQ
0 5 10 15 20 25 30
0 5 10 15 20 25 30
0 5 10 15 20 25 30
NOISE 
3.3 
10 
LIMIT OF DETECTION
LIMIT OF QUANTITATION
11 Proprietary & Confidential
Accuracy/Precision
Accurate/precise Not accurate/precise
Accurate/not precise Not accurate/not precise
12 Proprietary & Confidential
Precision
REPEATABILITY:
N x one analyst,
one apparatus
REPRODUCIBILITY:
N x more analysts,
more apparatus
13 Proprietary & Confidential
CAPILLARY
ELECTROPHORESIS
14 Proprietary & Confidential
1. Capillary electrophoresis– in general
2. On the field of separation techniques – strengths, weakness
3. Separation of inorganic ions – examples
4. Separation of polar and ionic substances – examples
5. Chiral separation – examples
15 Proprietary & Confidential
CE
+
+
+
+
-
-
+
-
-
++
+
+
Different velocity
(mobility) of ions
in el. field
+ -kV
capillary
detector
elektrolyt, elektrody
CE, CZE, HPCE
time
16 Proprietary & Confidential
CE - mobility
-
-
-
+
+
+
- +
+
+
-
Ev
= 
U
LL
t
L
U
t
L cd
c
d

 ==
),,,,,,( TIrpHpKQ  =
17 Proprietary & Confidential
CE – dissociation
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
oct[-10-9
m2
s-1
V-1
]
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
amm[10-9
m2
s-1
V-1
]
][
]][[
+
+−
=
HB
HB
Ka
ACETATE ION
AMMONIUM ION
18 Proprietary & Confidential
polymer
silica
cavity
EOF – (electro)endoosmotic flow  separation of
cations, anions, neutrals during one run
19 Proprietary & Confidential
CZE – capillary zone
electrophoresis
(M)EKC – (micellar)
electrokinetic chromatografy
CEC – capillary
electrochromatografy
20 Proprietary & Confidential
CZE – examples
0
5
10
15
20
25
30
2 3 4 5
time [min]
Abs[mAU]
E 122
E 110
E 124
Ltot = 48.5 cm
Ldet = 40.0 cm
pH = 2.2
U = -20 kV
2 ml susp. in 5 ml vol. flaskN
N
S
O
ONa
O
R1
R2
Test of dyes (E124, E110 a E122) in Ibalgin susp.
21 Proprietary & Confidential
 low running costs compared to GC a HPLC
 high separation efficiency
 fast analyses
 short method development
 worse robustness and inj. repeatability
 LOQ ~ 0,1 až 0,2 %
 Peak area depends on migration time
bubble cell a Z-cell
22 Proprietary & Confidential
electrolyte
pressure
1.
capillary
2.
3.
sample
Hydrodynamic
injection – the most
common in CE
23 Proprietary & Confidential
Where we can use CE and COMPLETE other separation techs:
• INORGANIC IONS
• IONIC ORGANICS
• CHIRAL SEPARATION
• BIOPHARMA
24 Proprietary & Confidential
CE examples
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7
čas [min]
A[mAU]
B1
B2
B6
B3
C
B9
B5
0
20
40
60
80
100
120
140
2 3 4 5 6
time [min]
Abs[mAU]
K
Na
Ca
Mg
Mn
Zn
25 Proprietary & Confidential
CE examples
Omega H3 Geriavit Pharmaton
component declared
value/cps.
CE* declared
value/cps.
CE*
C 60 mg ✓ 60 mg ✓
B1 20 mg ✓ 2 mg ✓
B2 5 mg ✓ 2 mg ✓
B6 10 mg ✓ 1 mg ✓
Nicotinamide 25 mg ✓ 15 mg ✓
Calcium pantothenate 10 mg ✓ 10 mg ✓
Mg(2+) 30 mg ✓ 10 mg ✓
Mn(2+) 2 mg not found 1 mg not found
Zn(2+) 15 mg ✓ 1 mg not found
26 Proprietary & Confidential
Chiral separation
EKC CEC
packed
Coated wall
monolithic
CD Proteins MIP
CSP
Chiral additives
Chirasil-DEX permethylated beta
CD
BSA lysozym cytochrom c
CE chiral separation
27 Proprietary & Confidential
CE chiral separation
a-CD b-CD g-CD
6, 7 a 8 glucopyranose units
selector analyte complex
• hydrophilic surface and hydrophobic inert part of cavity
• derivatisation => positive/negative charged selector
!
28 Proprietary & Confidential
28
CE chirální separace
Addition into
BGE
OT-CEC
packed and
monolithic
CEC
CE chiral separation
29 Proprietary & Confidential
A: silica capillary
B: „coated“ silica capillary (OT-CEC)
CE chiral separation
30 Proprietary & Confidential
30
0
50
100
150
200
250
0 1 2 3 4 5
time [min]
Abs[mAU]
(R)-N-Me-atomoxetin
(S)-N-Me-atomoxetin
0
50
100
150
200
250
0 1 2 3 4 5
time [min]
Abs[mAU]
(R)-N-Me-atomoxetin
CH3
O
N
CH3
CH3
CE chiral separation
31 Proprietary & Confidential
0
1
2
3
4
5
6
7
8
9
10
4 4.5 5 5.5 6
time [min]
mAU
(S)-duloxetine
(S)-N-Me-duloxetine(R)-duloxetine
(R)-N-Me-duloxetine
Limit of detection (LOD) and limit of quantitation (LOQ)
The limit of detection has been calculated by the formula:
S
σ
=
3.3
LOD
σ is ASTM noise of baseline value (0.138 mAU)
S is the slope of regression line - (11.0 mAU/%)
The detection limit of (R)-duloxetine has been 0.04 % on current condition
The quantitation limit is triple of this value, i.e. 0.12 %
CE chiral separation
32 Proprietary & Confidential
0
5
10
15
20
25
0 50 100 150 200
čas [s]
A[mAU] R-clopidogrel
S-clopidogrel
0
5
10
15
20
25
30
35
40
45
50
0 20 40 60 80 100
čas [s]
A[mAU]
R-citalopram
S-citalopram
+ -kV
capillary
detector
electrolyte, electrodes
CE chiral separation
33 Proprietary & Confidential
+ -kV
kapilára
detektor
elektrolyt, elektrody
CE biopharma
34 Proprietary & Confidential
CE biopharma
35 Proprietary & Confidential
CE biopharma
36 Proprietary & Confidential
MASS
SPECTROMETRY
37 Proprietary & Confidential
1. Mass spectrometry – in general
2. Qual/Quan approach on MS
3. Hyphenated MS (GC-MS, LC-MS)
4. Configuration of MS
5. Structural analysis in Pharmacy - examples
38 Proprietary & Confidential
MS úvod
MS – mass spectrometry
Analytical techniques that measures mass-to-charge ratio
39 Proprietary & Confidential
MS úvod - historie
lkgm =
J.J. Thompson, 1897
40 Proprietary & Confidential
intensity
m/z
Bvz
r
vm
=
 2
MS úvod
41 Proprietary & Confidential
1942: Calutron – separation of 235U a 238U
MS úvod
42 Proprietary & Confidential
„borrowing“ term SPECTRUM…SPECTROMETRY
Light dispersion – visible range – by the prism
43 Proprietary & Confidential
Dispersion with prism
- formation of elmg. spectrum
Separation of ions in mag. field
- formation of mass spectrum
44 Proprietary & Confidential
M M+
M1+
M2+
ionisation
fragmentation
m/z
m/z
M1+
M2+
M+
„hard“ ionisation
„soft“ ionisation
Ionisation
45 Proprietary & Confidential
M M+
M1+
M2+
ionisation
fragmentation
MSn
M1
+
M11
+
M12
+ MS/MS (MS2)
46 Proprietary & Confidential
Instrumentation
ION SOURCE ION
OPTICS ANALYZER + MULTIPLIER
TURBOMOLECULAR PUMPS
SAMPLE (MIXTURE)
SEPARATION (HPLC, GC, CE)
47 Proprietary & Confidential
Analyzers
0 50 100 150 200 250 300 350 400 450 500
0 5 10 15 20 25
FFT
čas [ms]
m/z
0 50 100 150 200 250 300 350 400 450 500
0 5 10 15 20 25
FFT
čas [ms]
m/z
48 Proprietary & Confidential
Resolution
465 467 469 471 473 475 477
m/z
C9H35O2N3B18Co
ORBITRAP
465 467 469 471 473 475 477
m/z
ION TRAP
RESOLUTION
49 Proprietary & Confidential
MS – Orbitrap
50 Proprietary & Confidential
291.98 292.00 292.02 292.04 292.06 292.08
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
292.04031
C10 H15 O5 N P S
292.02656
C8 H11 O3 N5 Cl S
15 000
Resolution
51 Proprietary & Confidential
291.98 292.00 292.02 292.04 292.06 292.08
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
292.04031
C10 H15 O5 N P S
292.02656
C8 H11 O3 N5 Cl S
20 000
Resolution
52 Proprietary & Confidential
291.98 292.00 292.02 292.04 292.06 292.08
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
292.04031
C10 H15 O5 N P S
292.02656
C8 H11 O3 N5 Cl S
25 000
Resolution
53 Proprietary & Confidential
291.98 292.00 292.02 292.04 292.06 292.08
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
292.04031
C10 H15 O5 N P S
292.02656
C8 H11 O3 N5 Cl S
35 000
Resolution
54 Proprietary & Confidential
291.98 292.00 292.02 292.04 292.06 292.08
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
292.04031
C10 H15 O5 N P S
292.02656
C8 H11 O3 N5 Cl S
50 000
Resolution
55 Proprietary & Confidential
291.98 292.00 292.02 292.04 292.06 292.08
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
292.04031
C10 H15 O5 N P S
292.02656
C8 H11 O3 N5 Cl S
100 000
Resolution
56 Proprietary & Confidential
Ion Routing
Multipole
C-Trap
HPC
LPC
MP0
Q1
Full OTMS scan
1st parent ion isolation in Q1
First parent trapped in the ion trap
Orbitrap
MS2 scan
m/z
Full OTMS Scan
m/z
UVPD MS/MS Scan
UVPD in the Ion Trap
MS – Orbitrap
57 Proprietary & Confidential
ION SOURCE ION
OPTICS ANALYZER + MULTIPLIER
TURBOMOLECULAR PUMPS
SAMPLE (MIXTURE)
SEPARATION (HPLC, GC, CE)
Hyphenated techniquesMS
58 Proprietary & Confidential
Hyphenated techniquesMS
MS
Determination
(QUANTITATIVE)
Identification
(QUALITATIVE)
10 15 20 25 30
čas [min]
UV
10 15 20 25 30
čas [min]
MS
HPLC-UV
HPLC-MS
ALMOTRIPTAN
10 15 20 25 30
čas [min]
UV
10 15 20 25 30
čas [min]
MS
HPLC-UV
HPLC-MS
ALMOTRIPTAN
59 Proprietary & Confidential
10 15 20 25 30
čas [min]
UV
10 15 20 25 30
čas [min]
MS
HPLC-UV
HPLC-MS
ALMOTRIPTAN
LC-MS identifikace
60 Proprietary & Confidential
Chromatography
STATIONARY
PHASE
MOBILE PHASE
61 Proprietary & Confidential
GC: gas chromatography
injection
detection
časseparation
62 Proprietary & Confidential
GC – typické příklady
Determination of
alcohol in blood
(headspace technique)
63 Proprietary & Confidential
GC-MS: gas chromatography – mass spectrometry
64 Proprietary & Confidential
GC-MS: gas chromatography – mass spectrometry
RK_RAM_62_run01 #1331 RT: 16.28 AV: 1 SB: 18 16.21-16.26 , 16.32-16.41 NL: 4.05E4
T: + c Full ms [ 50.00-600.00]
50 100 150 200 250 300 350
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
332.0
330.0
334.0
144.2
304.0
115.1
306.1
302.1116.2 225.1
251.2
329.1
143.2 172.2 335.087.1 89.2
injection
detection
separation
65 Proprietary & Confidential
GC – typické příklady
66 Proprietary & Confidential
Configuration HPLC
Autosampler
Column
PUMP AUTOSAMPLER DETECTOR
COLUMN
67 Proprietary & Confidential
HPLC
250 mm x 4.6 mm x 5 um
68 Proprietary & Confidential
nonpolar MP (org)polar MP (water based)
69 Proprietary & Confidential
70 Proprietary & Confidential
71 Proprietary & Confidential
Trends: U-HPLC
Ultra-High-Performance-Liquid-Chromatography
< 200 bar > 400 bar
72 Proprietary & Confidential
LC – moderní systémy
Pressure up to
1500 bar
ULTRA HPLC
U-HPLC
73 Proprietary & Confidential
ION CHROMATOGRAPHY
74 Proprietary & Confidential
75 Proprietary & Confidential
76 Proprietary & Confidential
77 Proprietary & Confidential
78 Proprietary & Confidential
2020 TRENDS
79
CO TO JE?
biopharmaceutical BIOLOGICAL biologic BIOLOGICAL
MEDICAL PRODUCTS biologické léčiva BIOPREPARÁTY
biopharma products biofarmaka biofarmaceutika BIOLOGICAL
80
CO TO JE? PROČ?
-UMAB
-XIMAB
-ZUMAB
-ERCEPT
81
ANALYTICKÉ VÝZVY
Malé
molekuly
Chemoterapeutika
Malé proteiny
Protilátky
mAb
Antibody-Drug
Conjugate
adalimumab
C6428H9912N1694O1987S46
82
ANALYTICKÉ VÝZVY
83
• Acclaim PepMap RSLC C18, 2 µm
• 75 µm ID x 50 cm
• 650 – 700 bar @ 270 nL/min
• Gradient: 3 – 50% B, 600 min
• e. Coli digest
1 000 píků, 10 000 peptidů
UHPLC analýza velkých molekul
84
Integrovaná kolona v iontovém zdroji
nanoViper
kolona
termostat
ZDV
HV konektor emiter
EasySpray
85
+ -kV
kapilára
detektor
elektrolyt, elektrody
Integrovaná kap. elektroforéza v iontovém zdroji
ZipChip
86
Integrovaná kap. elektroforéza v iontovém zdroji
87
Integrovaná kap. elektroforéza v iontovém zdroji
Electropherogram of intact infliximab:
0 2 4 6 8Time (min)
0
20
40
60
80
100
RelativeAbundance
2.6
4.53.5
0 Lys2 Lys
1 Lys
5700 5720 5740
m/z
0
50
100
0
50
100
RelativeAbundance
0
50
100
5722.8
5717.9
5713.0
zoom
z=26
Full MS spectra from Q Exactive HF-X MS with BioPharma Option
5000 6000
m/z
0
50
100
0
50
100
RelativeAbundance
0
50
100
5722.85510.8
5314.1 5951.7
5717.9
5506.2 5946.6
5309.5
5713.0
5941.45501.4
5305.0
z=26
z=25
z=27
z=28 RT 2.6 min
RT 3.5 min
RT 4.5 min
2 Lys
0 Lys
1 Lys
3 ng of sample injected
88
Integrovaný PaperSpray
ElectroSpray
PaperSpray
89
Integrovaný PaperSpray
PaperSpray
90
Extrahovatelné a louhovatelné látky (E&L)
91
Extrahovatelné a louhovatelné látky (E&L)
EXTRACTABLE
Chemické látky, které se
uvolňují ze zařízení nebo
obalu za laboratorních
podmínek extrakce.
Proces nesmí degradovat ani
deformovat materiál.
LEACHABLE
Chemické látky, které migrují
z výrobního procesu, balení
nebo transportu do lékové
formy za normálních
podmínek užívání. L jsou většinou podmnožinou E
92
Extrahovatelné a louhovatelné látky (E&L)
• Obal• Etiketa
• inkoust
• solvent
• lepidlo
• lak
• změkčovač
• lubricant
• pigment
• stabilizátor
• antioxidant
• monomery
• retardant
hoření
• Krabička
• sterilizační přísada
• konzervant
• potisk
93
Extrahovatelné a louhovatelné látky (E&L)
EXTRACTABLE LEACHABLE
Testuje se materiál Testuje se produkt
94
molekulová hmotnost
polarita
HPLC – kapalinová
chromatografie
GC – plynová
chromatografie
HPIC – iontová
chromatografie
Analytická separace
Zvyšující se výskyt kontaminantů
95
Cílená a necílená analýza
cílená
kvantifikace
OČEKÁVANÉ
1x
Full-scan
Rozsah
necílený
screening
NEOČEKÁVANÉ
cílený
screening
PODEZŘELÉ
96
• LC-ICP-MS
• IC-ICP-MS
• ICP-MS
• ICP-OES
• ESI & APCI
• LC-UV or CAD
• LC-MS/MS HRAM
• IC-MS
• Library
• GC-MS
• GC-MS/MS HRAM
• EI & CI
• Library
• GC-MS
• GC-HRMS
• Headspace
• EI & CI
• Library
Analýza
97
Accelerated
solvent
extraction
technology
Preparation
MS certified
vials, columns
and ultra-pure
solvents
Consumables
Headspace
sampling and
compliant
GC-MS
Volatiles
Advanced
Orbitrapbased
HRAM
GC-MS/MS
Semi-volatiles
Advanced
Orbitrapbased
HRAM
LC-MS/MS
Non-Volatiles
Robust,
compliant
ICP-MS
Elemental
EXTRAKCE ANALÝZA A REPORTOVÁNÍ
Komplexní řešení pro E&L
98
• Analogické k USP 467
• Molekuly jsou obecně známé a
jednoduše kvantifikovatelné
Vzorek + valve &
loop headspace
Robustní
separace
Identifikace a
kvantifikace pomocí MS
Compliant data
processing
HS plastové fólie
Těkavé látky
99
Thermo Scientific™ TriPlus™ 300 headspace sampler
Thermo Scientific™ Trace 1310 GC
Thermo Scientific™ TraceGOLD TG-624SilMS GC
Columns
Thermo Scientific™ ISQ™ Series GC-MS
Thermo Scientific™ Dionex™ Chromeleon™ CDS
Thermo Scientific™ AppsLab Library™
Těkavé látky
100
Netěkavé
Netěkavé látky
101
• Detekce v pozitivním i negativním módu
• Elementární složení
• Informace o struktuře (fragmentace)
• Zachytit vše – kombinace s Corona CAD
Extrakt Separace a
detekce všeho
Identifikace s
HRAM MS/MS
Software
Netěkavé látky
102
Data from ESA Biosciences, Inc., Chelmsford, MA
BLANK
Netěkavé látky
103
Data from ESA Biosciences, Inc., Chelmsford, MA
BLANK
VAK 1
Netěkavé látky
104
Data from ESA Biosciences, Inc., Chelmsford, MA
BLANK
VAK 1
VAK 2
Netěkavé látky
120 nalezených látek
105
Kovy
Prvková analýza
106
• Analogické k ICH Q3D a USP 232 & 233
• Stopová analýza kovů, tolerance k org. matrici
• Thermo Scientific™ iCAP™ RQ ICP-MS
Extrakt Automatická příprava
vzorků
ICP-MS Compliant data
processing
Prvková analýza
• Stříbro (Ag) se uvolnilo ze 4 vaků
• Olovo (Pb) se uvolnilo z 28 vaků
107
Semi-volatiles
Odpařitelné, semi-volatilní
108
• EI/CI ionizace, full scan HRMS 120k
• Možnost MS/MS
• Excelentní SW podpora
• Thermo Scientific™ Q Exactive™ GC
system
Extrakt Separace Identifikace s HRAM
MS/MS
Software
Odpařitelné, semi-volatilní
109
Identifikace
C15H16O2
CH3 CH3
OH
OH
OH
Q Exactive GC MS Parameters
Transfer line (°C): 280
Ionization type: EI
Ion source(°C): 230
Electron energy (eV): 70
Acquisition mode: Full scan
Mass range (Da): 50-700
Mass resolution (FWHM): 120k
Lockmass (m/z): 207.03235
TRACE 1310 GC Parameters
Injection Volume (uL): 1
Liner Single gooseneck
Inlet (°C): 280
Carrier Gas, (mL/min): He, 1.3
Oven Temperature Program:
Temperature 1 (oC): 40
Hold Time (min): 1
Temperature 2 (oC): 320
Rate (oC/min) 5.5
Hold Time (min): 12
Q Exactive GC Hybrid QuadrupoleOrbitrap
GC-MS/MS system
Trace 1310 GC system
TraceFinder software
110
Identifikace
C15H16O2
OH
Vzorky těsnění
• A červené
• B hnědé
• C bílé
• D černé
• Blank
Plechovka
• 300 ml hexanu
• 16 hod/lab. teplota
• Zakoncentrování na 1 ml
111
Identifikace
961 látek!
112
Identifikace
• detekcí píků
• HR
dekonvoluce
• „čištění“ spektra
Detekce/dekonvoluce
1
Generování kandidátů
2
Filtrování, identifikace
3
• hledání v
knihovnách
• generování
kandidátů
• HR filtrování
kandidátů
• hypotéza,
identifikace
preferován (semi)automatizovaný proces
113
Identifikace
Kombinace SI (search index) a HRF (high resolution filtering) = hodnota skóre (%)
∑ (m/z *Intenzita)vysvětlené
∑ (m/z *Intenzita)nalezené
HRF skóre = x 100%
114
Identifikace
C8H8O2
115
Identifikace
116
Identifikace
měření
M+2 izotopový klastr
117
Identifikace – přehled
118
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