high productivity in rapid resolution lc with triple

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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

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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?

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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

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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

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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)

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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

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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

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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

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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

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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

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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.


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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

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Software – Compliance Features

Identify user: ConfigurableRole-based permissions

Quant Audit trail: Configurable Logging and Presentation

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Software – Compliance Features

FDA Auditor

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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

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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)

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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.

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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

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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*

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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

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Results – Chromatographic Performance

x1000 Amoxicillin

Terbutalin (ISTD)Clavulanic acid

1.3 min

PWHH = 0.03 min

PWHH = 0.02 minPWHH = 0.015 min

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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

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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

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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

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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

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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.

high productivity in rapid resolution lc with triple

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