High Productivity in Rapid Resolution LC with Triple ...
Transcript of High Productivity in Rapid Resolution LC with Triple ...
Page 1 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
High Productivity in Pharmacokinetic and
Bio-availability Studies
Rapid Resolution LC with Triple-Quadrupole MS detection and MassHunter software
Plas
ma
conc
entra
tion
Time
Page 2 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Introduction
Is the researcher interested in peak areas? In concentrations of a drug compound in a single sample?
Probably not!
AUC∞
CmaxF
CL
The researchers are interested in parameters like:
• What is the Area under the curve? • What is the maximum plasma concentration of the drug?• After what time is the maximum concentration reached?• What is the absorption rate?• What is the elimination rate?• What is the bio-available of the drug?
Page 3 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Introduction
Typical form of a plasma level curve and certain calculated parameters of interest.
Pla
sma
conc
entra
tion
Time
Absorption phase Elimination phase
Plasma level curveCmax
tmax
(Minimal therapeutic concentration)
(toxic concentration)
Area under the curve (AUC)C1/2
t1/2
Therapeutic range
Elimination Rate kElim
Page 4 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
LC-MS/MS System - Overview
Agilent High throughput QQQ-RRLC-System
• 2 Binary Pumps SL• CTC HTC-Pal autosampler with active wash station and cooled stack• Thermostatted Column Compartement SL with 2ps/10pt valve for Alternating
Column Regeneration• 2 Agilent Zorbax RRHT SB C18 2.1mm x 50 mm, 1.8µm, resp. Agilent Zorbax
RR SB C18 2.1mm x 50 mm, 3.5µm columns• 6410 Triple Quadrupol MS• MassHunter Software for Acquisition, Qualitative and Quantitative Data Analysis
Page 5 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – MassHunter Quantitative analysis
Sample batch
Target chromatogram(Quantifier/Qualifier)Clavulanic acid
ISTD chromatogram(Quantifier/Qualifier)
Calibration curve
Problem flagging(in this case too low response because of blank sample)
Target Quantifier Results
ISTDQuantifier Results
(Additional columns available for all available results)
Page 6 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – MassHunter Quantitative analysis
Method set-up tasks
Sample Information(TIC of all compounds)
Compound Information(Target, ISTD; Quantifier,Qualifier)
MRM information for Quantifier, Qualifier, ISTD information, etc. directly pulled from acquisition method!
(Additional columns available for all available tasks)
Method set-up in two minutes
Page 7 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software Overview – The Power of Excel-based reporting
The data-flow in MassHunter
Raw Data
MassHunterQuantitative
Analysis
Excel™Worksheet and print-out with Study-Results
Excel™ Template
Import sample data &Do custom calculations
Results File
Page 8 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – The Power of Excel-based ReportingMassHunter uses Microsoft Excel to generate reports.
An Excel template has to be created once. Then, during reporting this template imports automatically all data from the batch results file and does user defined calculations.
The power of this: All calculation and formatting (incl. conditional formatting!) functionalities of Excel are available !
Data import area of the template
Custom calculations
Page 9 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – The Power of Excel-based Reporting
Typical PK parameters calculated directlyduring MassHunter report generation
The use of a 3rd party Excel add-in - PK Functions* for Microsoft Excel – providing formulas to calculate PK typical parameters demonstrated here to calculate AUC, AUC∞, Cmax, Tmax, kEl, T1/2.
*) developed by Joel I. Usansky, Atul Desai and Diane Tang-Liu, Department of Pharmacokinetics and Drug Metabolism Allergan, Irvine, CA 92606, U.S.A.
Individual sheet per patient/volunteer
Page 10 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software - The Power of Excel-based ReportingCustomized Reports with the PK values directly available after analysis:
Results for the individual sampling times of one patient
Calculated pharmacokinetic parameters of one patient
Patient-ID
Page 11 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – Compliance Features, the ATM
QuantAdministrator
A QuantAdministrator has to set the permissions for the different MassHunter commands, allow / allow with reason / deny for the different user levels:
e.g. removing a sample from a batch is only allowed for users of the QuantBatchAnalyzer-group. To do so, an individual user validation is mandatory and a reason.
Increasing productivity of clinical pharmacokinetics
Month ##, 200XPage 12
Logical Flow of the Audit Trail Map:
Does ATM require password
or reason?
Is user in required Role?
Audit Trail record created
Action permitted
Action not permittedNo
User attempts an action
User enters requir-ed information
Users/ Group
ATM
Audit Trail record created
AuditTrail
No
Yes
Yes
Page 13 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – Compliance Features
Identify user: ConfigurableRole-based permissions
Quant Audit trail: Configurable Logging and Presentation
Page 14 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Software – Compliance Features
FDA Auditor
Page 15 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Study - Overview
This study covered the high throughput analysis of plasma samples containing a combination drug composed of Amoxicillin and Clavulanic Acid, commercially available under the trade name AUGMENTIN® (contains 250mg Amoxicillin and 125mg Potassium clavulanat).
Amoxicillin Potassium Clavulanat
Page 16 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Study - Principal
LC-MS/MS Analysis6
1 Drug Application 2 Blood sampling after certain time points
4 Calibration samples in blank plasma
Contr
0min 40min 60min20min 8 h
Matrix
3 Blood workup to yield plasma samples
Add anticoagulant (citric acid or EDTA) to blood, centrifuge to yield plasma
Sample Workup for LC-MS/MS Analysis5
(Details on next slide)
Page 17 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Assay – Plasma Workup details
Because of the high polarity of the compounds a back-extraction of the acetonitrile used for the protein precipitation was required.
Evaporation of the acetonitrile and reconstitution of the solution was not advised because of the known sensitivity of clavulanic acid.
Page 18 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Assay – Plasma Workup details
LC-MS/MS Analysis6
3 Back-extraction of Acetonitrile
300µL CH2Cl2
5 Repeat 3 and 4, transfer to autosampler vials/MTP
1 Protein Precipitation
200µL Acetonitrile with 0.517ng/µL ISTD
100µL Plasma Sample
2 Centrifugation and transfer of supernatant
10min @15000 rpm
4 Centrifugation and transfer of aqueous phase
5min @10000 rpm
Page 19 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Method - HPLCSolvent A: Water (0.005% HOAc)Solvent B: Acetonitrile (0.005% HOAc)Flow: 0.8 ml/minTemperature: 40 °CValve: Switch to nextInjection volume: 4µL
*) System overhead time will be longer if method changes!
% B
Eluent pump
Regeneration pump(in backflush mode)
1
30
% B
1
95
0.9 1.1 1.300.5
HTC Pal Autosampler
1.50Time [min]
0.08
Draw, Inject, Clean
ca. 0.5 min
System O
verhead Time
min. 0.15m
in*
Page 20 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Method - MS
General: Time Filter: OffDeltaEMV: 800
Source: Ion Source: ESIDGasHeater: 250 °CDGasFlow: 11 L/minNebulizer Pressure: 50 psiVCap: 4000 V
MRM-Settings: MS1Res: UnitMS2Res: UnitDwell time: 30 ms
0.00 - 0.341060136108198.1negClavulanic Acid
0.34 - 1.3020120125152226.2posTerbutaline (ISTD)
0.34 - 1.3010120208114366.2posAmoxicillin
TimeSeg[min]
Collision Energy [V]
Fragmentor [V]
Qualifier Product Ion
Quantifier Product Ion
Precursor Ion
PolarityCompound
Page 21 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Results – Chromatographic Performance
x1000 Amoxicillin
Terbutalin (ISTD)Clavulanic acid
1.3 min
PWHH = 0.03 min
PWHH = 0.02 minPWHH = 0.015 min
Page 22 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Results – Comparison 1.8µm vs. 3.5µm
The RRHT columns produce ca. 1.3 times narrower peaks than the RR columns, but with a higher backpressure and a higher potential to clogging, especially with these demanding matrices. Back-flushing during column wash and re-equilibration with the second pump was used to minimize clogging.
Finally the user has to select whether to spend more effort on workup to prevent clogging or to change columns more often if he would like to take the resolution and sensitivity advantage of sub-two-micron columns.
Terbutalin peak of the 1ng/µL calibration solution:
Abundance vs. Acquisition Time (min)0.62 0.64 0.66 0.68 0.7 0.72 0.74 0.76 0.78 0.8 0.82 0.84
6x10
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 1.8 µm, w5σ = 0.03min
3.5 µm, w5σ = 0.04 min
Page 23 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Results – Calibration curves
Amoxicillin
9 Levels, 9 Levels Used, 27 Points, 25 Points Used, 0 QCs
Relative Concentration-0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Rel
ativ
e R
espo
nses
y = 0.2057 * x
R2 = 0.99989794
LLOQ: 10.1 pg/µL Amoxicillin in Plasma
ULOQ: (not determined)
Range: 0.010 ng/µL 10 ng/µL
Abundance vs. Acquisition Time (min)
x104
0
0.25
0.50
0.75
1.00
1.25
1.50
1.75
0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2
10 pg/µL
20 pg/µL
50 pg/µL
100 pg/µL
Page 24 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Results – Calibration curves
Clavulanic Acid
5 Levels, 5 Levels Used, 15 Points, 13 Points Used, 0 QCs
Relative Concentration
0 1 2 3 4 5 6 7 8 9 10 11 12 13
-4x10
-0.250
0.250.5
0.751
1.251.5
1.752
2.252.5
2.753
3.253.5
3.754
4.254.5
4.75
Rel
ativ
e R
espo
nses
y = 4.1936E-005 * x - 2.7931E-007
R2 = 0.99474559
LLOQ: 0.05 ng/µL Clavulanic Acid in Plasma
ULOQ: (not determined)
Range: 0.05 ng/µL 5.0 ng/µL
x103
0
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
Abundance vs. Acquisition Time (min)
0.05 0.1 0.15 0.2 0.25 0.3 0.35
0.05 ng/µL0.25 ng/µL0.50 ng/µL
2.50 ng/µL
5.00 ng/µL
Page 25 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Results – Plasma concentrations
µg*h/mLµg/(mL*h)hµg/mLµg*h/mL
4.24±14.66AUC ∞
0.03±0.41kElim
0.18±1.00Tmax
1.33±4.00Cmax
3.30±14.17AUC
µg*h/mLµg/(mL*h)hµg/mLµg*h/mL
1.27±6.60AUC ∞
0.06±0.39kElim
0.38±1.00Tmax
1.35±2.57Cmax
1.17±5.98AUC
Amoxicilline Clavulanic Acid
Amoxicilline / Clavulanic Acid
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00Time [h]
Con
cent
ratio
n [µ
g/m
L]
Clav_AverageClav_1
Clav_2
Clav_3
Clav_4
Clav_5
Amox_AverageAmox_1
Amox_2
Amox_3
Amox_4
Amox_5
Page 26 Month ##, 200X
Increasing productivity of clinical pharmacokinetics
Summary
The Agilent 6410 TripleQuad LC-MS/MS System together with the 1200 Series Rapid Resolution LC system offers a very high productivity by:
• Parallel column operation to reduce the overall cycle-time.
• Easy and straight forward software operation.
• Direct reporting of pharmacokinetic relevant values and not onlyindividual concentration values by fully customizable Excel reports.
• Direct generation of Excel files for any further processing, documentation etc. (no csv-file export/import or file conversion actions).
• Software designed to comply with the regulations.