2. Overview
The impact on Bioanalysis of modern Drug
Discovery
Di
The Stages of Bioanalytical Process
Sample Collection
Sample Preparation
LC/MS/MS
Removing bottlenecks
Addressing bioanalytical challenges
3. Bioanalysis in Drug Discovery
= analysis in a biological matrix
quantitative analysis Chem. measurement of the
amounts of the constituents of a substance
in vivo activities in vitro activities
Pharmacokinetics (drug
Ph ki ti Metabolic Stability
M t b li St bilit
concentration vs time) Inhibition
Brain penetration (B/B ratio,
ti + Cell Permeability
single time point or time course)
Plasma Protein Binding
PK/PD (effect vs Other
concentration)
4. Discovery Bioanalysis
Bioanalytical Advantages Bioanalytical Challenges
GLP Validation not required GLP Validation not required
No need for LTS Must quantify compounds in a rapid
Smaller sample numbers screening environment
Medium-sensitivity assay Simple sample processing
Minimum assay development Rapid chromatography
In-house samples collection with Need generic assay for analysis to
rapid processing allow for rapid screening of multiple
compounds
Single IS for multiple compounds
Multiple matrices (in vitro/in vivo)
High specificity for the compounds of
interest
5. The Impact on Bioanalysis
To produce fast and simple method development
To produce rapid acquisition of high quality data for critical
decisions
To produce quantitative determination of drug,
metabolites,
metabolites at sub-ng/mL levels
6. …1997
HPLC -MS/MS
Standard sample
preparation • Sensitivity (
y (Low
ng/mL)
40uL plasma
• Specificity
• Selectivity
Add 100uL CH3CN
•SSpeedd
+ Internal standard
• RT 2-10 min
Vortex
Method development
time: 1-2 days
Centrifuge
…to date
UPLC -MS/MS
Transfer to microvials
• Sensitivity (pg/mL)
Analyze by HPLC- • Specificity
API/MS/MS • Selectivity
• Speed
• RT 1-2 min
Method development
time: hours?
7. …however
biological fluids are complex matrices that contain
numerous different compounds, ranging from simple
inorganic salts to large proteins.
Although matrix components remain undetected because
of the selectivity of the MS/MS detection, they can affect
the quantitation of analytes (Bonfiglio R. et al. Rapid Communic.
q y ( g p
Mass Spectrom. 1999)
8. Discovery Bioanalysis – tipping the balance
DISCOVERY
Bioanalysis
Throughput Q
Quality
y
CHEMISTRY BIOLOGICAL
BIOLOGY MATRICES
…how?
9. Stages of Bioanalytical Process
Improve Throughput reducing bottlenecks
Automation
sponse
Fast Methods
(and method x
Res
Dosing developments)
x
x
compound X x
Sample Conc.
Collection
C ll ti
Data Processing
Automation and
Sample Instrument
preparation optimization
Chromatography LC-MS interface Mass spectrometry
10. Stages of Bioanalytical Process
Improve Quality addressing bioanalytical challenges
Stability
y
Matrix Effect Sensitivity and
Selectivity
sponse
x
Res
Dosing x
x
compound X x
Sample Sensitivity and
Sample
p Conc.
Collection
C ll ti Selectivity
volumes Data Processing
Sample
Carry-over preparation Matrix Effect
Chromatography LC-MS interface Mass spectrometry
11. Work-Flow for Quantitative LC/MS/MS
Data Review
and Reports
Sample Sample LC MS/MS
Collection preparation separation Analysis
Many aspects of a bioanalytical method focus on the performance
of a method as it is used in the analytical laboratory. However, other
procedural nonanalytical elements can also affect the actual, or
apparent,
apparent measured amount of the analyte in the sample
sample.
12. Sample Collection and Storage
Sample
storage
Dosing
compound X
Sample
Sample collection
preparation
Handling and Storage of the Some analytes are not stable
samples prior to analysis can result
under standard sample collection
in a change in the amount of
analyte in a sample
conditions
If an analyte is less stable in whole blood
e.g. than in plasma, any delay in processing
the sample or poor temperature control
could result in analyte loss.
13. Inherent instability results from:
enzymes localized in the blood and non-enzymatic process e g :
non- process, e.g.:
tissues of all species, e.g.:
Blood pH-dependent instability
Butyrylesterases Shift in pH of biological fluids
Acetylcholinesterases
Tissues
Carboxylesterases –several
C b l t l
isoforms If the samples contain pH-labile
compounds the pH shift during
Species differences in esterases: sample preparation could affect
–Rat > dog > monkey > human the analysis results
14. Change of pH of ex vivo rat plasma under different
conditions at 37 C with time
37°
9 8.8
8.5 7.46 7.68 7.55
pH 8
Physiological
7.5
7 pH of Plasma: 7.35 - 7.45
6.5
6
Rat plasma 10% CO2 Citrate buffer Phosphate buffer
The shift of plasma p from the p y
p pH physiological value can affect important
g p
phenomena pH-dependent
Chemical stability of compounds
Protein binding
A. Fura, J.
A Fura J of Pharmaceutical and Biom Analysis Vol 32
Biom. Analysis, Vol. 32,
Issue 3, 14 July 03
14
15. Analyte Stability in Discovery Bioanalysis
General Approach Special handling & stabilization
needed during collection and
In-house collection with rapid
I h ll ti ith id processing & analysis:
i l i
processing:
instability addressed by NaF to inhibit esterases
addition of acetonitrile Buffer used to lower pH to
followed by rapid stabilize metabolites (e.g. acyl-
centrifugation
g g
glucuronides))
No need for LTS Perform all work on ice to ensure
no additional degradation
16. Equilibrium Dialysis Using Biological Matrices
BEFORE INCUBATION AFTER INCUBATION
INCUBATION
5h 37ºC
37 C
Matrix spiked with compound Buffer
Membrane (12-14 KDa)
Dilution 1:1 with Dilution 1:1 with
drug-free dialysed buffer drug-free dialysed matrix
(Matrix-Buffer)
%Bound = * 100 Protein precipitation, centrifugation and dilution of supernatant
Matrix
%Unbound = 100 - %Bound
17. Impact of Stability on Brain Tissue Binding
t=0 t= 5h t=0 t= 5h
α-Conotoxin MII 100
% Remaining cmpd
80
Gly Cys Cys Ser
83%
60
g
Asn Pro Val Cys His
Leu Glu His
40
Ser Asn Leu Cys NH2 20 0.15%
0
Determination of free
drug concentration w/o protease inhibitor with protease inhibitor
w/o protease inhibitor with
protease inhibitor
in Lister Hooded
Lister-Hooded
rat brain
homogenates by
equilibrium dialysis
%Bound = <50 %Bound = 92.1
%Unbound = >50 %Unbound = 7.9
18. Case Example: Bioanalytical Challenge
1200
y = 0.0005x + 11.297 7.E+05
1000 R² = 0 9536
0.9536 6.E+05
Analyte Area
800 5.E+05
Analyte/IS
Blood samples 600
4.E+05
3.E+05
collected from Dog 400 2.E+05
1.E+05
-62%
200
and stored at -20°C
d t d t 20°C 0
0.E+00
QC 200 fresh QC 200 F/T
0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06
120 120
emaining cmpd
maining cmpd
100 100
80 80
60 60
40 40
% Rem
% Re
20 20
0 0
DOG RAT H2O/ACN APCI 1:3 Dilution
Fresh I F/T cycle II F/T cycle Fresh I F/T cycle
Freeze-Thaw instability, matrix and species-dependent
(not a matrix effect)
19. Case Example: Bioanalytical Challenge (cont’d)
I F/T
Fresh II F/T
120
pd
100
% Remaining cmp
80 Issue addressed through
60 HCOOH or HEPES buffer
40 added
R
20
upon samples collection
0
H2O 0.1% HCOOH HEPES
Fresh I F/T cycle II F/T cycle
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) is a zwitterionic organic
chemical buffering agent. HEPES is widely used in cell culture, largely because tests
indicate it is better at maintaining physiological pH despite changes in carbon dioxide
when compared to bicarbonate buffers. HEPES is a preferred buffering agent for
maintaining enzyme structure and function at low temperatures
20. Take Home Messages
Bioanalytical methods for inherently unstable
compounds are best developed keeping biological and
p p p g g
chemical instabilities in mind
Bioanalytical results -even though the assay is
validated-
validated are only as good as the collection and
processing procedures…
21. Work-Flow for Quantitative LC/MS/MS
Reports
Sample
from in Sample Analysis
LC/MS/MS
house preparation and data
animals review
23. LC/MS/MS –Good News, Bad News
Good news: You only see what you want
to see
Bad news: What you don’t want to see....
is still there (Sometimes in the current
run; sometimes in later runs)
24. Animal species and Major
matrices commonly
ti l Components of
C t f
used: Biological Fluids
Species Matrices
Rat Blood Salts
Mouse Plasma Proteins, peptides
,p p
Dog Brain Phospholipids
Gerbil Liver Other lipids (e.g., fatty
Guinea Pig acids)
id )
Marmoset Unknowns...
Cynomolgus
C l
25. Common Extraction Techniques
Protein precipitation Fast,
Fast easy –limited selectivity
limited
Liquid/liquid extraction Application specific,
Application-specific improved selectivity
Solid phase extraction
p Application-specific, Highly selective
pp p , g y
On-line techniques Application-specific, carryover
Turbulent flow
On-line SPE
Valve-switching
26. Protein Precipitation
Solvent Solvent Volume Residual (mg)
Many practitioners in drug
ACN 2x
2 4.30
4 30
discovery do this – 3X excess 3x
4x
0.46
0.54
acetonitrile, mix, centrifuge, MeOH 2x 7.14
3x 3.36
(
(blow down/recon), inject
), j 4x 3.95
Pros: Cons:
•Simple, fast and automated •Non-selective extraction
•Universal •Risk of matrix suppression
•Can provide “clean” samples for
Can clean •Not as rugged as other sample
Not
some compounds preparation methods
•Suited for discovery work •Results can be erroneous
•For development work, stable
For without stable isotope IS
isotope IS is required
27. What is Matrix effect?
Is the ff t f
I th effect of co-eluting, undetected matrix component on
l ti d t t d ti t
the ionization of the target analyte
The competition between co-eluting compounds and the
analyte of interest may reduce or enhance the ion intensity of
the analyte affecting the reproducibility and the accuracy of
the assay. (Matuszewsky et al. Analytical Chemistry,2003)
28. Implications
The matrix effect problem must be evaluated to ensure
p
reliable quantitation of analytes and assure the integrity
of pharmacokinetic data
The efficiency and reproducibility of the ionisation
process is affected leading to erroneous quantification
results - Pharmacokinetic data are compromise -
Loss in sensitivity - Matrix effects cause severe
problems in methods at low pg
p pg/mL in bioanalytical
y
matrices -
29. How to study? 1- Direct Comparison
Standard Solution
Response Standard
p
LC-MS/MS
LC MS/MS
solution
Mobile phase
Spike standards
p
Blank sample matrix into extracted matrix
Extraction Response Post-
LC-MS/MS extracted spiked
sample
Blank matrix Post-extracted
spiked sample
%Matrix effects = (Response Post-extracted spiked sample-1) x 100
Response Standard solution
Negative value = suppression
Positive value = enhancement
30. How to study? 2 - Post-column infusion
10µl blank mobile phase – MRM analyte
XIC of +MRM (1 pair): 476.0/220.0 amu from Sample 3 (plasma intero mouse) of 5HT1abd_pos... Max. 1.2e4 cps.
10µl blank plasma extract – MRM analyte 1.7e4
1.6e4
AUTOSAMPLER Blank mouse plasma
1.5e4 Analyte injected
injected
COLUMN 1.4e4
1.3e4
1.43
1.2e4
T- PIECE 1.1e4
1.0e4
1.40
9000.0
8000.0 0.02 1.10
1.07 1.31
7000.0 0.93 1.49
6000.0
5000.0 0.89
ANALYTE
4000.0
HPLC PUMP flow
0.69
3000.0 1.62 1.72 1.84
0.99
600µl/min 2000.0 0.08
1000.0
0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time, min
INFUSION PUMP 10 µL/min
(1-10ng/mL)
3 – Incurred samples
-Dilution
-Increasing the i j ti volumes
I i th injection l
31. Protein Precipitation: Matrix Effect
Dilution
plasma diluted 1:6 plasma diluted 1: 21,6
MATRIX %M S M ATRIX %M S
GW compound
p Plasm a DOG -36
36 Plasm a DOG -20
20
Plasm a GERBIL -31 Plasm a GERBIL -17
Plasm a RAT 34 Plasm a RAT 17
n=6 n=6
FLUOXETINE plasma diluted 1:6 plasma diluted 1: 21,6
MATRIX %MS MATRIX %MS
F F Plasma GUINEA PIG -48 Plasma GUINEA PIG 4
Plasma MOUSE -44 Plasma MOUSE 4
F
Plasma RAT -35 Plasma RAT 9
O N
n=6 n=6
plasma diluted 1:6 plasma diluted 1: 21,6
SB compound MATRIX %MS MATRIX %MS
Plasm a MOUSE
as OUS 37 Plasma MOUSE 13
Plasm a RAT 26 Plasma RAT 18
n=6 n=6
Dilution can reduce matrix effect
32. Automation for Sample Prep. Systems
Why What
Increase throughput
Costs vs. benefits
Improve quality of data
greater performance consistency
over time Complexity vs real needs
more reliable method transfer risks of errors
i k f
Improve safety difficult to control and check
Eliminate tedious work the process
(avoid potential human mistakes)
33. Automated Sample Prep. Procedure
1. Preparation of Calibration Standards
Automated serial dil ti of a t standard i control
A t t d i l dilution f top t d d in t l
matrix
Automated serial dilution of a top standard in aqueous
solutions and spike of equal volume into control matrix
2. Extraction and dilution
-200µL sample + 400 µ AcN
µ p µL Extraction
Centrifugation
-100µL supernatant + 80µL H2O Dilution
-(or blow down/reconstitution)
34. Case Example: Bioanalytical Challenge
Rat Blood Calibration Curve
4500
cs1 0.005 385 R² = 0.6507
4000
cs2 0.01 2575
3500
cs3 0.02 680
3000
Analyte Area
cs4 0.04 1033
2500
cs5 0.1 2165
2000
cs6 0.2 3940
1500
cs7 0.4
04 7355
1000
cs8 1 20200
500
cs9 2 39200
0
cs10 4 84300
0 0.05 0.1 0.15 0.2 0.25
cs11 10 224500
Conc
Calibration Curve in H2O/ACN
C r e
20-fold dilution
in Blank Matrix
Calibration Curve in Matrix
Fixed Tips
36. Tissue Sample Preparation: current method
Manual weighed
brain tissue in Homogenization
15mL plastic tubes using Autogizer®
Manual dilution
a ua d u o
with MeOH:H2O Manual aliquots
(1:1) of blank and
unknown samples
37. Tissue Sample Preparation: Bioanalytical Challenge
5’24” homogenization
following 1hr incubation
with collagenase
g
Muscle Manageable by RSPs!
15’ homogenization
Not manageable by RSPs
38. Preparation of Calibration Standards (CS)
and QC samples
CS and QC are prepared by spiking a biological matrix with analytical
standard solutions. The biological matrix used must be from the same
solutions
species and strain as the study samples. If this matrix is difficult to
obtain, a surrogate matrix (another species) may be used, however the
validity of the surrogate matrix should be verified if possible by
verified, possible,
comparing responses from at least one spike in each matrix
The control matrix should be harvested using similar procedures e g
procedures, e.g.
type of anti-coagulant, to that to be employed for the Study samples to
be analyzed
Ideally calibration standards should be prepared on the day of dose
administration and stored with the study samples
Where feasible, the percentage of non-matrix solvent present in
calibration standards or QC samples should be less than 5% (v/v), with
no more than 2.5% (v/v) organic solvent
39. Case Study: Using Fresh or Frozen Blood for
Calibration and QC samples
XIC of +MRM (4 pairs): 442.1/342.0 amu from Sample 1 (rat1, PO, 2h) of GHS_PK_001_prove_... Max. 941.3 cps
941
900
0.99
0 99
Parent
850
(Rat1, 2hrs, PO) MRM 442/342
800
750
700
650 “Metabolite” “Likely”
Likely
600
550 MRM 428/159 formation of
“Metabolite” N-desmethyly
500
450
MRM 428/342
400
350
300
metabolite
250
200
150
100
50
0
0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40
Time, min
Analysis of Blood and Brain Samples from a Standard
PK Study in Rat
40. Case Study (cont’d): CS, QC sample preparation
XIC of +MR M (5 pairs): 442.1/159.0 am u from S am ple 1 (cs11 1430) of G H S_P K0 01_RTB L_BE ... Max. 2.4e5 cps
0.94
2.4e5
On the day of the dose, rat drug-
y , g
2.2e5
2.0e5
20 5 Blood Parent
free diluted blood and brain 1.8e5
Calibration 442/159
homogenates were thawed,
1.6e5
1.4e5
Standard (356000 cps)
spiked with working solutions 1.2e5
and f
d freezed again at -20°C
d i t 20°C
1.0e5
8.0e4
(48h). 6.0e4
4.0e4
2.0e4
0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
T im e, min
XIC of +MRM (5 pairs): 428.0/159.0 amu from Sample 1 (cs11 1430) of GHS_PK001_RTBL_B... Max. 4302.7 cps.
Metabolite
0.94
“Metabolite”
4200
4000
at 1 / 2%
3800
428/159
3600
3400
On the day of the analysis, study 3200
parent in
samples, CS and QC were thawed,
3000
2800 (6010 cps)
2600
2400 all CS
processed as usual and analyzed. 2200
2000
1800
1600
samples
1400
1200
1000
800
600
400
200
0
0.2
02 0.4
04 0.6
06 0.8
08 1.0
10 1.2
12 1.4
14 1.6
16 1.8
18 2.0
20
Time, min
41. Case Study (cont’d): Why is the “Metabolite”
Present in Blood Calibration Curves ?
XIC of +MRM (5 pairs): 428.0/159.0 amu from Sample 1 (cs11) of GHS_PK001_RTBR_BEP160... Max. 105.9 cps
0.92
105
Impurity?
100
95
“Metabolite”
90
85
NO! M t b lit detected in blood CS
Metabolite d t t d i bl d 80
75 428/159 Brain
B i
(150 cps) Calibration
70
but not in brain CS and in pseudo 65
60
CS samples
55
50
Standard
45
40
35 0.73
0.96
30
25
20
0.84 1.25 1.37
15
10
Interference from matrix? 5
0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Time, min
NO! Drug free blood is blank
Drug-free
Instability of parent compound in fresh
blood?
Instability of parent compound in extracted NO! M t b lit /P
Metabolite/Parent ratio is constant
t ti i t t
matrix? (ca. 0.05%) in CS prepared with fresh
NO! Metabolite/Parent ratio is constant (ca. diluted blood and stored at room
0.05%) in extracted matrix at least for 4 temperature for 24 h
p
days at 4°C
42. Case Study (cont’d): Studying the problem...
Metabolite Parent Metabolite/Parent
Peak Area (cps) Peak Area (cps) Ratio
Spike in fresh
diluted blood 6
stored at 4 °C for 692
Parent 1.62·10
1.62 10 0.05%
4 days 442/342
Spike in fresh
diluted blood 6
stored at -20 °C 4740 1.59·10
1.59 10 0.3%
for 4 da s
days
Spike in thawed
diluted blood 6
stored at -20 °C 14446 1.55·10
1.55 10 1%
for days
f 4d
Sample integrity in blood was compromised by freezing
(thawed blood seems to be different from fresh blood)
43. Preparation of CS and QC samples: Surrogate Matrices
Premise: Blank matrices may be obtained:
Externally: some as blank matrices (e.g. for PK
studies) may be difficult to obtain (and may be
very expensive)
In-house: each animal submitted to a withdrawal
(g
(generally 1 mL p PK study), can’t be subjected to
y per y), j
any in-vivo studies for the following weeks (as
expressed by international guideline).
44. Surrogate Matrices: diluting with Rat Blood
Curve preparation:
190µL of fresh diluted rat blood (1:4), spike of 10µL of
working solutions
Sample preparation:
1) Dilution 1:5
) 160µL fresh diluted rat
µ
blood (1:4) and 40µL of sample
2) Dilution 1:10 180µL fresh diluted rat
blood ( ) and 20µL of sample
(1:4) f
45. Diluting with Rat Blood: Results
268_Blood Concentration AB172_Blood Concentration
ng/mL)
g/mL)
Study Concentration
Study Concentration 400
150 Dilution 1:10
Blo Concentration (n
Bloo Concentration (ng
Dilution 1 10
Dil i 1:10
Dilution 1:5 300 Dilution 1:5
100
200
50 100
ood
od
0
0
0 2 4 6 8
0 2 4 6 8
Time (h)
Time (h)
RESPONSE FACTOR
0.0200 RA T
87_Blood Concentration MA RMOSET
ation (ng/mL)
Study Concentration 0.0150
100
Dilution 1:10
75 Dilution 1:5
RF
0.0100
Blood Concentra
50
0.0050
25
0
0.0000
0 2 4 6 8 1 2 3 4 5 6 7 8 9 10 11
CS
Time (h)
Opportunity to save valuable matrix and in particular possibility
to increase studies in vivo (no delay caused by animal rest)
in-vivo
46. Work-Flow for Quantitative LC/MS/MS
Reports
Sample
from in Sample Analysis
LC/MS/MS
house preparation
ti and data
animals review
46
47. LC/MS/MS Method: Introduction
The development of an LC/MS/MS method requires two separate
methodologies to be developed:
Chromatography. To separate the analyte(s) of interest from
both endogenous interferences and drug metabolites
MRM MS. To confirm resolution of the compounds of interest
from endogenous metabolites or other interferences
47
48. Autosamplers
•Capacity 96 well- and 384 well-plates
•Automation
•Shortest Cycle Times (injection speed, overlapped injections
(< 30sec)
•Lowest Carry Over (inside and outside needle wash and injector
programming (< 0.003%)
48
49. Carryover
Carryover can cause quantitation errors in a low concentration
extract which follows the injection of a high concentration
extract
Carryover is estimated using peak area ratios (response).
The response of a matrix blank fortified with internal standard ( ) only, that is injected after
p (IS) y, j
an upper limit of quantitation (ULQ) extract is compared to the analyte response in an
LLQ extract and expressed as a percentage of the LLQ.
Carryover: = < 20%
49
50. Carryover: Troubleshooting
Column Autosampler
1. Increase Organic Flush of 1. Classify carryover (classic or constant)
Analytical Column 2. Replace blank
2. Change column or Use 3. Change injection size
Monolithic columns
M lithi l 4. Check fitting assembly
3. Reduce curve range 5. Check wash solvent
a. Use fresh wash solvent
b.
b Increase wash volume
c. Use more organic solvent in wash
Other d. Adjust wash pH
6. Check washing mechanism
1. Use HN ion source (less 7. Change injection solvent
sensitive) 8. Check another sample
2. g
Reduce curve range 9. Change hardware
a. Change needle seal
b. Change injection loop
c. Rebuild or replace valve
50
51. Carryover: Strategy for Sample Analysis
Matrix blanks assayed following high standard and high
q
quality control ( ) samples.
y (QC) p
QCs and CSs assayed in ascending concentration order.
Study samples assayed in PK profile
profile.
Do troubleshooting
51
52. LC Method development
LC methods should meet the following criteria:
Accuracyy Q
Quantification limit
Precision Linearity
Specificity Range
Detection limit Robustness
52
53. LC Method development Workflow
Screen for Analyte Determining the retention characteristics of the
Retention compound(s) of interest
Select pH
Select Organic Move the retention of the analyte of interest towards
Modifier the end of the chromatographic analysis to prevent
interferences from metabolites
Optimize the
Gradient
Test in Matrix Test the analyte in matrix
Check Specificity by calculating the matrix
y g MS/MS Method
effect and assess
Check Sensitivity interferences
53
54. Modes of HPLC
Normal phase & aqueous normal phase (HILIC)
Reversed phase
Ion
I exchange
h
Size exclusion
Affinity chromatography
Reversed phase HPLC is by far the most widely used
54
55. Benefits of reversed phase HPLC
Reversed phase HPLC allows the use of binding
p g
interactions ranging from hydrophobic bonding to ionic
interactions, offering a large range of selectivities
The use of a more polar mobile phase compared to the
stationary phase often means the use of primarily
aqueous mobile phases which are safer to use and easier
to dispose of than the primarily organic mobile phases of
normal phase HPLC
Compatible with aqueous samples
Most d
M t drug metabolites are more polar th th d
t b lit l than the drugs th
they
derive from, therefore, the metabolites elute first.
55
56. Why Choose Gradient Elution
To separate samples having Cons
components that vary widely in
p y y Re equilibration
Re-equilibration time
polarity (= generic) adds to analysis time
To separate low molecular Instruments vary in their
weight mixtures having large dwell volume (Vdw), which
number of components (= high can cause method transfer
separating power) problems
56
57. To Maximise Gradient Resolution Between Peaks
N
Rs = 4 ( α -1
α ) ( k
k+1 )
System Selectivity Retentivity
Efficiency
Increase one or more of the following:
k* Gradient retention Change the chemistry of the
mobile or stationary phase
Change % organic
α Selectivity Change the chemistry of the
mobile or stationary phase
Change % organic
N Theoretical Plates Decrease particle size or flow
rate
Increase column length
57
58. All of the Following Increase Gradient Retention (k*)
• A longer gradient time tG Fast G di t Ch
F t Gradient Chromatography
t h
• A shorten column Vm This means that the column can be
• A higher flow rate F shortened and the flow rate increased
(within pressure constraints) and the
• A shorten organic range ∆Ф gradient time can then be reduced
without loss of separating p
p g power.
Because 1/ k* ∝ Gradient steepness = b = (S ∆Ф Vm)/tG F
If “b” is kept constant from run-to-run peaks will elute
i k t t tf t k ill l t
in the same relative pattern
58
59. Speeding Up Analysis: Flow Rate Column Length
OH-midazolam
Reserpine GW
Fast gradient 50x2 mm, 5µm
OH-bupropion
column, flow 0.8mL/min,
Paracetamol tg2.5min
Run time 3.8 min
GW
OH-midazolam
Faster gradient 30x2 mm,
Reserpine
OH-bupropion 4µm column, flow
Paracetamol 1.5mL/min, tg1.3min
Run time 2.3 min
Reserpine
OH-midazolam GW
OH-bupropion Faster gradient 20x2 mm,
Paracetamol 3µm column, flow
3 l fl
2mL/min, tg0.8min
Run time 1.8
59
60. Always Consider MATRIX EFFECT
Tg and Flow rate Flow 1.5mL/min, Tg 1.3min
tg 0.8
flow Area H20 Area rat Ion supp% Area dog Ion supp%
1 4.45 1.29 71 1.35 70
1,2
, 4.3 1.66 61 1.52 65
1,5 3.85 1.58 59 2.01 48
tg 1,3
13
flow Area H20 Area rat Ion supp% Area dog Ion supp%
1 4.57 2.22 51 2.84 38
1,2 4.16 2.68 36 3.7 11
1,5 3.54 2.89 18 3.61 0
Example: 1OH Mid
E l 1OH-Midazolam, Z b SB C-18 30X2 3 5 µm
l Zorbax C 18 3,5
60
61. Ultra Performance Liquid Chromatography
What is it?
Similar to HPLC
Silica based stationary phases
Same mobile phases
Same chromatographic principles we are used to
Similar pumps / autosamplers
Different to HPLC
Smaller particle size (<2 μm vs >3 μm)
Increased backpressure
61
62. Particle Size
Column efficiency (N) is proportional to column length &
y( ) p p g
inversely proportional to the particle size
L
N∝
dp
Therefore the column can be shortened by the same
factor as a decrease in particle size with no loss in N
Evolution NOT Revolution
62
63. Separation Efficiency of Columns with
Different Particle Sizes
Diff t P ti l Si
Optimal velocity range
63
64. UPLC: Why are we interested?
Improved chromatographic resolution
Improved separation from other analytes
Improved separation from endogenous components (MS
suppression)
i )
Same resolution in less time
Increased sample throughput / productivity
Increased sensitivity
Narrower peaks are taller
But:
Requires a system which can sustain very high pressure
15000 psi vs. 6000 psi limit on Agilent 1100 pump
64
66. Optimized set-up: Reducing Time for Method Development
Few Generic Fast Gradients with two set of mobile phases
T(min) %A %B A: H2O, 0.1% HCOOH T(min) %A %B A: NH4HCO3 10mM pH=9.5
0 100 0 B: ACN, 0.1% HCOOH 0 90 10 B: ACN
0.2 100 0 Flow rate: 1500µL/min 0.3 90 10 Flow rate: 1000µL/min
1.5 5 95 1.0 5 95
1.7 5 95 A: CH3COONH4 5mM, 5%ACN 1.5 5 95
1.8 100 0 B: ACN 1.6 90 10
2.1 100 0 Flow rate: 1500µL/min 2.0 90 10
Since the majority of pharmaceutical analytes are ionic or ionizable
in nature, variation of the mobile phase pH induce larger changes in
, p p g g
selectivity than traditional solvent changes, in reversed-phase
chromatography
66
68. Dual LC-MS/MS Instrumentation
Column Selector
HP1100 with Stream selector
twin pumps and pumps and
columns columns
68
69. Avoiding Matrix Effect
XIC of +MRM (1 pair): 310.0/44.0 amu from Sample 2 (plasma intero mouse) of 5HT1abd_postc... Max. 4.8e4 cps.
4.8e4
0.02 In th
I the END zone late
l t
By increasing k’ and 4.5e4
Eluent diversion eluting matrix components
avoids all the non- can affect analyte
providing more 4.0e4
retained interferences ionisation (e.g.
retention time of from entering to mass
g phospholipids)
1.75
analytes, the 3.5e4
spec SAFE 1.57 1.72
1.82
“unseen” 3.0e4
WINDOW 1.53
1.43
interferences from 2.5e4 1.27
matrix are mostly
separated from 2.0e4 FRONT zone
FRONT zone
0.92
analytes eliminating
0.90
1.5e4
the ion suppression. 1.0e4
0.09
5000.0 END zone
0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time, min
(Matuszewski at al Anal Chem 1998;Fu Woolf Matuszewski J Pharm
al., Anal. Chem. 1998;Fu, Woolf, Matuszewski, J. Pharm.
Biomed. Anal, 1998; Murphy et al., Rapid Commun. Mass Spectrom., 2002)
69
70. Valve switching
Stream selector
POSITION A MS and waste
PUMP 1
WASTE
POSITION B
1
2 MS Source
10 PUMP 1
WASTE
3
9 MS Source
1
8 10 2
4
7 PUMP 2 3
9
8
4 PUMP 2
WASTE
7
COLUMN
WASTE
70
71. Phospholipids: why are they an issue?
O O
R2 O HO
O R1 O R1
O m/z184 + 2H
O O
O P O P
m/z104 + H
O O O O
Polar
Head Group
+ +
N N
R1, R2 = C12-C18
, Lysophosphatidylcholine
Phosphatidylcholine
Phosphatid lcholine
Ubiquitous (present in all species)
High-levels
High levels (mg/ml in plasma)
Strongly retained (accumulate/bleed)
Surfactants
Matrix-variable (
M ti i bl (e.g., h
human di t )
diets)
Unstable –degrade to fatty acids
Recent phospholipid-based matrix effects presentations and publications on www.tandemlabs.com/capabilities/
71
publications.
72. Phospholipids: The impact
XIC of +MRM (11 pairs): 322.0/116.0 amu from Sample 1 (cs11 2860 ng/mL) of TRUI_PK014_GSK1360707_po_DOBD_hua121006_01...
4.8e6
m/z496 Max. 7.9e4 cps.
4.6e6
4.4e6
4.2e6
4.0e6 m/z524
3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
Intensity, cps
2.8e6
2.6e6
2.4e6
s
2.2e6
2.0e6
1.8e6
1.6e6 Analytes m/z704, 758, 786
1.4e6
1.2e6
1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
0.88
0.0 0.98 1.04 1.16 1.81
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Time, min
The presence of phospholipids in extracted samples can result in:
retention time shifts
elevated baselines
divergent curves
signal suppression/enhancement
72
73. Phospholipids: The impact
Analyte
Analyte Analyte 2nd injection
1st
injection 65%-75% gradient
65%-75% gradient
Phospholipids
-late eluting-
3rd injection 4th injection
65%-75% -100% 65%-75% -100%
gradient gradient
Analyte
Anal te Analyte
Phospholipids
-late eluting-
Phospholipids
Chromatographic Improvements and Overcoming Matrix Effects and Carryover for a Previously Validated Nine Analyte LC/MS/MS Assay Using
UPLC. Edmonda Cook, Min Meng, Patrick Bennett Tandem Labs, Salt Lake City, UT 73
74. Handling Phospholipids
Dioctyl Phthalate 391:149
Lyso-Phoshatidylcholine (16) 496:184
MRM monitoring for phospholipids during Lyso-Phoshatidylcholine (18) 524:184
method development m/z 496, 524, 704, Phoshatidylcholine (30) 704:184
758, 786, 806 Product Ion m/z 184 (or
( Phoshatidylcholine (34) 758:184
Precursor of m/z 184) Phoshatidylcholine (36) 786:184
Phoshatidylcholine (38) 806:184
Determine if chromatographic separation of phospholipids occurs
Back or forward column flush
Post-column
Post column infusion of extracts with injection of analyte
Select APCI over ESI whenever possible
Improve sample preparation:
SPE, L/L – specific conditions required
74
75. Injection Factors
The eluotropic strength of the diluent and the injection volume significantly affect
chromatographic efficiency. Ideally, a diluent that is weaker than the mobile
phase should b employed. If a stronger dil
h h ld be l d t diluent i unavoidable, i j ti
t is id bl injection
volume is best kept low.
ACN 50% ACN 37%
ACN 37% ACN 50%
ACN 50%
75
76. MS Method development
The parameters that must be determined for
successful MS detection and quantification are:
Ionization mode
MRM transition ions
77. The LC-MS Interface
Dr. John Fenn
Nobel Lecture
Atmospheric Pressure Ionisation
p
77
