Automation and Validation of HPLC

Linda A. Brunner
February 1999

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 Objectives

 Automated pharmaceutical HPLC analyses

- Direct injection from microplates
 Combinatorial analysis, high-throughput screening and
pharmacokinetic assays
- Validating method linearity using automated serial dilution
- Automated derivatization
 Validation aspects of HPLC systems and analyses
- Structural validation at the manufacturing level
- On-site instrument validation and calibration (IQ/OQ, IPV)
- Method validation and SST software
- Holistic system validation

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Automation using an X-Y-Z Autosampler

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 Types of Laboratory Automation

 Fixed geometry - fixed programming

 Fixed geometry - flexible programming
 Flexible geometry - flexible programming

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 Series 200 Autosampler
Optimized HPLC sampling automation
 Non-pneumatic, random access
- Sample injection volume: 0.1-2000 µL
- Fast sampling (25 sec per injection)
 High precision and flexibility
- RSD: < 0.5% (3-100 µL)
- Linearity: 2-100 µL (r2 > 0.999)
- Carryover: < 0.02%
 Vial-to-vial transfer and mixing
 Sampling from 10+ optional sampling trays
- Standard vials, micro-vials, microtiter trays (standard and
deep-well), Peltier-cooled

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Fast Injection for HTS

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 Combinatorial Chemistry
Lead generation and optimization

Parallel synthesis
Microplate
in 96-well, deep
microplates
Manual

Series 200 Autosampler

LC purification
LC/MS analysis
and identification

Auto

Microtiter Plate Tray

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 Series 200 Micro Plate Handler
Extended micro plate handling capacity
 Robotics accessory for
transport of either standard or
deep-well microtiter plates
 Capacity:
- 72 standard
- 20 deep
 Total samples/injections:
1,920 to 6,912
 Control:
- Series 200 Autosampler
firmware
- PE Sciex LC/MS
workstation

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 Automation in Discovery and Research

 Applications
I. Automated identification by generation of extracted ion
chromatograms and mass spectra of combinatorial
mixtures
II. On-line column switching to isolate active peptides for
LC/MS characterization
III. Rapid quantitation of drugs and metabolites in serum,
plasma or tissue samples

Note: All analyses were performed using the PE Series 200 autosampler,
Series 200 micro pumps, and PE Sciex API 150EX LC/MS, or API 2000 or
API 3000 LC/MS/MS systems

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I. LC/MS Screening of Combinatorial Mixtures
Automatic generation of XICs for identification
TIC from IB4-15 3.88e6 cps

LC/MS TIC

3.2e6

2.4e6

1.6e6
Intensity, cps

8.0e5

1 2 3 4 5 6 7 8 9
534.2 Time, min 518.2
1.1e5
XIC from 518.2 and 534.2 amu from IB4-15 2e4 5.00e5 cps
518.2
XIC 534.2 & 518.2

4e5

3e5

379.2
2e5 376.2
Intensity, cps

716.0 754.4
1e5 600 600
m/z, amu 534.2 m/z, amu

1 2 3 4 5 6 7 8 9
Time, min

Courtesy of Dr. Dan Kassel, CombiChem using the CombiSyn™ system

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 Combinatorial Library Analysis
XIC’s for all expected synthetic products
Masses read 2.5e7

in from text 2.0e7

file...
1.5e7
576.4
618.4 1.0e7
Intensity, cps

632.6
5.0e6
666.5
675.6
10 15 20 25 30 35 40
682.6 Time, min

705.6
708.6 4.0e6 731.6
717.6 632.6 705.6 682.6 811.6 781.6
3.2e6 666.5 772.6
722.6
722.6 618.4 708.6 675.6 747.6 761.6
576.4
731.6 2.4e6

747.6 717.6
1.6e6
Intensity, cps

761.6
772.6 8.0e5

781.6
811.6 10 15 20 25 30 35 40
Time, min

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 II. Screening Combinatorial Libraries
Column switching to isolate active peptides for LC/MS

M3, M 4 and
Unbound M 1 & M 2

Receptor-Ligand
Complex
Denatured
R
R R - M1 SEC due to LC
R G-25 Mobile Phase Short
R R - M2 R Conditions DVB Column
M1 R -M 1 (Step Gradient) RR
R RM
R-M 2
M3 M3
M2 Time M1 2 Previously
M4 M4 Bound M1 & M 2
& Free Receptor

Switching Valve
MS
LC or MS/MS

Courtesy of S. Kaur, et al, 43rd ASMS, Atlanta, GA., May 1995

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 Characterization of the Active Peptide
Peptides before and after receptor activity screening
731.6
632.6 705.6 682.6 772.6 811.6 781.6
666.5 708.6 722.6
4.0e6 618.4 675.6 747.6 761.6
576.4
3.2e6 Before 717.6
Intensity, cps

2.4e6

1.6e6

8.0e5

10 15 20 25 30 35 40
Time, min

4.0e6 747.6
After
3.2e6
Intensity, cps

2.4e6

1.6e6

8.0e5

10 15 20 25 30 35 40
Time, min

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III. Quantitative Pharmacokinetic Analysis
Bosentan metabolites in dog bile

IonSpray/MS
1.0e5

250 300 350 400 450 500 550 600 m/z

1.0e8 LC/MS TIC

Intensity 3 6 9 12 15 18 21 24
568 R.T. (min.)
538
1000 IonSpray/MS/MS 552

250 300 350 400 450 500 550 600 m/z

6e5
LC/MS/MS TIC

3 6 9 12 15 18 21 24 R.T. (min.)

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 Linearity and Sensitivity
Bosentan metabolites in dog bile
7
Conditions: Flow injection analysis; SRM; 5µL injected into 200µL/min

50 pg y = 0.929x + 4.383 r 2 = 0.996

300
6 Log Area
Intensity

200

5
5 pg
100 Log Height
Blanks
y = 0.893x + 3.885 r 2 = 0.995
Log Response

(3)
4 1 2 3
N+ H2
H+
CH2 O O
N S
time (min) 4.2e4
202.0
N
B
D
NH O
N O O
A N
N B C
N
N O
3 A

Intensity
N OH

280.1 552.2
2
311.1
255.0 508.2

200 250 300 350 400 450 500 550

m/z
1
(3) (2) (1) 0 1 2 3

Log Amount (Nanograms)

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Automated Standard Curve Generation

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 Automated Method Linearity Validation

 Method linearity is often validated manually

- Preparation and injection of a series of standard solutions
 Series 200 Autosampler can automate validation
- Injections of increasing volumes from a single calibrant
- Performing automated serial dilution and calibration

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 Increasing Volumes of Single Calibrant
Data based on 10 injections at each volume (analyte: t-butyl benzene)
C u r v e P a r a m e te r s :

C u rv e #1: 1st O rd e r
W e i g h ti n g F a c t o r = 1. 0 r 2 = 0. 9 9 9 8 9 7
8 x 1 07

6 x 1 07
1 0 0  L, 0. 1 3 % R S D
A r ea

4 x 1 07
2  L, 0. 3 9 % R S D

5 0  L, 0. 1 5 % R S D
R2 = 0.999897
5  L, 0. 2 9 % R S D
2 x 1 07

2 5  L, 0. 1 4 % R S D

1 0  L, 0. 1 7 % R S D

0
0. 0 2 0. 0 4 0. 0 6 0. 0 8 0. 0 1 0 0. 0 1 2 0. 0 1 4 0. 0

In je c t e d Vo lu m e

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 Automated Serial Dilution

 Standard is placed in first vial

 Empty vials are placed at
positions 2-5
 Programmed dilution
performed using the diluent
tank and appropriate
standards
 Injection of samples and
calibrants starts automatically

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Linearity and Precision Results

(4 replicates each)

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Automated Sample Preparation

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 Automated Derivatization

 Derivatization is a common technique to enhance the

chromatography or detectability of target analytes
 Common sample analytes are
- Fatty, organic and amino acids
- Alcohols, aldehydes, ketones, thiols, and amines
- Proteins, peptides and carbohydrates
- Drugs, steroids, pesticides, phenols and nitrosamines
 Application example
- Analysis of essential amino acids using precolumn OPA
derivatization

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 Analysis of Essential Amino Acids
Auto-dilution and derivatization with OPA

Protein Hydrolyzate Column: Pecosphere 3 x 3 C18

(3 µm, 33 x 4.6 mm ID)
Mobile Phase: A: Methanol
B: 1.5% THF in 50 mM
NaOAc (pH 5.9)
Linear gradient of 5% A to 80%
A in 6.5 min
Flow Rate: 2.5 mL/min
Detection: 340 nm (Ex) / 450 nm (Em)

Precision: < 2.0% RSD

Linearity: r2 > 0.9998 (1-200 picomoles)

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Validation: Instrumentation and
Methodology

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Validation of HPLC Systems and Analyses
What is Validation?
 Validation is
(as defined by the FDA’s 1987 Guideline on Process
Validation) “...establishing documented evidence which
provides a high degree of assurance that a specific
process will consistently produce a product meeting its
predetermined specifications and quality attributes.”

 Ultimate responsibility of the end user / sponsor

- Vendor can offer tools, documents and services to help

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Regulations: What is GLP, GMP and GALP?

 Good Laboratory Practice (GLP)

- Non-clinical laboratory studies regulations for
pharmaceutical development
 Proposed (1976), published (1978), revised (1987)
 Good Manufacturing Practice (GMP)
- Regulations for the manufacturing of pharmaceuticals
(and other products regulated by US FDA)
 Good Automated Laboratory Practices (GALP)
- Principles / guidelines to ensure integrity and traceability of
computerized data [Published by US EPA (1995)]
 Compliance is mandatory for pharmaceutical laboratories

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 System Validation Timeline

Vendor ’s Site User’s Site

Structural Functional Validation Re-calibration
Validation and Maintenance

Qualification

Installation Operational Performance

IQ OQ PQ
Before Use During Use

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Perkin-Elmer Validation Tools and Services

 Structural Validation
- Quality system for product development / manufacturing
- Product final test
 Instrument Validation Services and Documentation
- On-Site IQ/OQ and Instrument Performance Verification
(IPV)
- Documentation
 Certificate of System Conformity, IQ/OQ, SOPs
 PE Nelson structural validation and regulatory compliance
manual
 Software for Method Validation

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Perkin-Elmer Product Development Process

 ISO 9001 certified manufacturing sites

- British Standards Institution (BSI)
 Product Development Process includes:
- Marketing survey to define product feature sets
- Product proposal and specifications
- Test plan and engineering verification
- Alpha, beta and QA testing
- Documented product release
Note: Documents available for inspection with signed
nondisclosure agreement and a corporate signed
validation requirement procedure

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 Validation Tools: Internal Diagnostics

 Diagnostics and system verification tools

- Full operating and service diagnostics built-in along with
software to test RAM, ROM, keypad, display
- Pump: Power-up diagnostics, pump use log
- Detector: Lamp energy, lamp use log, wavelength
verification
- Autosampler: Power-up alignment and tray/vial detection

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 Product Final Test and Calibration

 Pre-shipment final test

- Pumps (3-hr test)
 Safety verification and electrical functional check
 Pressure, flow and composition calibration
 Leak test, 7-step passivation
- Detector
 Lamp alignment, wavelength calibration, 24-hour burn-in
 Beam energy, low angle scatter and spectrum check
 ASTM noise and drift test, ASTM linearity
- Autosampler
 Functional and leak tests, x-y-z alignment
 Precision, carryover, linearity

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 PE Nelson Software Development

 System Development Life Cycle (SDLC) Process

- Conforms to ISO 9001 (TICKIT) and FDA / EPA
guidelines for computer systems
 Steps
- Requirement analysis, functional specifications
- Design and Test Plan
- Implementation and Programming
- System Integration and Testing (SQA)
- Documentation and System Release
- Maintenance, Upgrade and Version Control

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 PE Nelson Site Audit Program

 Customer-conducted audit of PE Nelson

- Hosted > 100 audits
 Customer review of PE Nelson’s Quality System:
- Software development
- Testing and release procedures
- Product support and validation programs
- Other issues: configuration management, documentation
control, security, escrow and personnel training

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 Perkin-Elmer Product Support

 Operating manual shipped with all instruments

- Describes operation, maintenance, troubleshooting and
spare parts
 Technical support
- Field product specialists and in-house experts
- Support hotlines
- Technical support E-Mail and fax
 Service support
- Installation, warranty, IQ/OQ, IPV, and repair
 Training
- Specialists on-site or at PE technical centers

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 IQ/OQ Validation

 Installation Qualification (IQ)

- Procedure to verify and document that the instrument is
installed according to manufacturer's specifications and
directions
 Operational Qualification (OQ)
- Procedure to verify and document that the instrument
operates according to established specifications and
standards
 IQ/OQ is performed by the vendor, in-house metrology
group (or end-user)

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IQ: Installation Verification of each Module

 Instrument functional description

- Specific instrument ID (i.e. serial number), location
- Software / firmware revision
- Certificates and manuals
 Installation/configuration procedures
- Instrument specifications
- Site requirements (physical & electrical)
- List of supplies and consumables
- Safety precautions
 User approval completion
- Signature and date

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OQ: Operational Qualification of each Module

 Standard operating procedure (SOP)

 Certificate of Conformity
- Results of final test of the unit
 Functional validation procedure
- (i.e. PE’s IPV procedure, test results and certification)
 Calibration / maintenance schedule and logs
 Verification of operator training
 User approval of completion
- Signature and date

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 IPV Procedures, Service and Certification

 Instrument Performance Verification (IPV)

Verification of key operating parameters for each module
after installation or re-calibration
- Pump
 Flow and compositional accuracy, pressure pulsation
- Detector
 Wavelength accuracy, noise and drift
- Autosampler
 Precision and carry-over
- NCI Interface
 Accuracy of gain and linearity

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Interface Validation Module (IVM) and Software

 Analog peak generator for validating A/D interface

- Generates 8 analog patterns
- Calibrated and traceable to NIST reference voltage
 Interface validation software
- Automated 10-min verification test
 Checks voltage accuracy/gain error, precision, and linearity
- Generates validation test results and pass/fail report

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 Additional Programs from PE Nelson

 User manuals - electronic version

 Performance Qualification (PQ) assistance
 SQA test suite package(s)
 Source code escrow service

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 Perkin-Elmer Installation Services

 Standard installation includes:

- Pre-installation and Installation Checklist
- Abbreviated IPV
- Customer Orientation Script (familiarization)
 IQ/OQ installation includes the standard installation,
plus:
- Complete IPV testing and certification
- Complete documentation and sign-off
This is a requirement for a pharmaceutical laboratory

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 Validation: What is PQ?

PQ is “performance qualification” . . .
Validation that the entire analytical system functions
properly, generating data meeting established criteria

 PQ is a series of chromatography tests with SST criteria

- Linearity, accuracy, precision, sensitivity, specificity . . .
 PQ protocols are developed by the user, according to
their specific application(s) and SOPs
- PE has developed a rapid, holistic PQ protocol for Turbo
LC Plus systems using a universal test mix

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Method Validation: SST, Re-calibration, etc.

 Analytical method must be validated to obtain valid

sample data
- Includes sample preparation, chemicals / reagents, etc.
- Equivalency test after a parameter change (new column)
 Other considerations for valid sample data
SST - Before actual sample analysis, daily check of entire
system for compliance to system suitability parameters
(might include standard curves and QA samples)
Each module should be re-calibrated/validated at regular
intervals (e.g. 6-12 months) and after repair or relocation
The operator must have adequate credentials and training

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 USP Guidelines for Method Validation

Analytical Performance Category II

Parameter Category I Quant. Limit Category III

Precision Yes Yes No Yes

Accuracy Yes Yes ? ?
Limit of Detection Yes No Yes ?
Limit of Quantitation Yes Yes No ?
Selectivity Yes Yes Yes ?
Range Yes Yes ? ?
Linearity Yes Yes No ?
Ruggedness Yes Yes Yes Yes

Note: Category I = Major Component

Category II = Minor Component
Category III = Performance

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 Software Tools to Help with Method Validation

Turbochrom Tools / Features

Accuracy Interactive Graphic Method Editor
Precision Peak Summary
Limits of detection System Suitability (S/N Calculation)
Specificity m/Peak Purity Reporting, TurboScan,
System Suitability (n, Rs, Tf)
Linearity/Range Multilevel calibration, calibration plot ( r2)
IVM - Interface Testing
Ruggedness Turbo Method Development

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 Turbochrom Peak Summary
Automatic calculation of tR and peak area precision
** Summary Report ** .
Iso-Propyl Benzene Anthracene
File Sample Time Area Time Area
Name Name [min] [µV·s] [min] [µV·s]

5ul003 5 ul injection 0.84 1332838 1.03 1111930

5ul004 5 ul injection 0.82 1330947 1.03 1108623
5ul005 5 ul injection 0.84 1331692 1.02 1111280
5ul006 5 ul injection 0.84 1328473 1.02 1108039
5ul007 5 ul injection 0.84 1319670 1.02 1100541
5ul008 5 ul injection 0.84 1326515 1.02 1106971
5ul009 5 ul injection 0.83 1325539 1.02 1105507
5ul010 5 ul injection 0.84 1328437 1.02 1108216

Averages 0.84 1328014 1.02 1107639

%RSD 0.12 0.32 0.18 0.32

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System Suitability Testing

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Automated S/N Calculation: LOD Determination

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 Automatic Reporting
Wavelength maximum (max) and peak purity (PI)
TEST REPORT
Pk # Component RT Area Height
1 Comp A 0.437 3016138 1389.5432
2 Comp B 0.633 5049419 1970.0206
3 Comp C 0.890 4342001 1361.9135
4 Comp D 1.203 3550203 904.0825
5 Comp E 1.600 3209537 653.6164
6 Comp F 2.213 6287826 954.6478

PEAK # TIME Wavelength MAX PI

1 0.44 254 nm 4.51
2 0.63 260 nm 3.05
3 0.89 260 nm 1.40
4 1.20 259 nm 1.21
5 1.60 258 nm 1.06
6 2.21 250 nm 1.17

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 What are FDA inspectors looking for?

 Documented personnel credentials

 Standard operating procedures (SOPs)
 Validated analytical method(s)
 Accessible raw data
 Security of data system and data back-up
 Evidence of analytical systems being properly installed,
operated, maintained and calibrated

They want you to document what it is that you do, do it as

is/was documented, and document that it was done!

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Holistic Validation

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Holistic Approach to HPLC System Validation

 “In a holistic validation approach, a series of tests are used

to measure and evaluate the performance of the entire
computerized LC system under the conditions of its
intended use - chemical analyses.”

 Furman1, WB, Layloff1, TP, and Tetzlaff1, RF. “Validation of Computerized

Liquid Chromatographic Systems”. Presented at the 106TH AOAC Annual
International Meeting and Exposition, Workshop on Antibiotics and Drugs in
Feeds, August 30, 1992, Cincinnati, OH.

 1Food and Drug Administration, Division of Drug Analysis, 1114 Market

Street, Room 1002, St. Louis, MO 63101.

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 Holistic HPLC Testing Procedure

 Fast, simple gradient separation providing:

- tR Precision (flow rate/compositional accuracy)
- Peak area precision (autosampler, integrator, or detector)
- Sample carryover (injector problems)
- Absorbance maxima (detector wavelength calibration)
- Concentration linearity (detector, injector)
- Signal-to-noise ratio (detector sensitivity)

Reference: “A Holistic approach for system validation”, PE Applications, 1995.

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Holistic PQ Protocol for Turbo LC Plus Systems

 Automated Sequence of 13 Injections

Standard: Universal test mix diluted 1/1 in 35/65 ACN/Water
Column: PE Brownlee RA C18 3x3 column, N = 4000 plates
Gradient: 35-95% ACN in water, linear over 6 min, hold at 95% for
1 min, equilibration at 35% for 3 min
Flow rate: 1.5 mL/min

Parameter Inj Vol # Injections Run Time

evaluated (L) (min)
Reproducibility 10 6 60
Carryover 10, “0” 2 20
Linearity, WL 3, 5, 20, 30, 50 5 50
Calibration
Total 13 130 min

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 Holistic Validation: Reproducibility
Area RSD for t-butyl benzene = 0.498%, RSD for tR = 0.028%

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Holistic Validation: Carryover

Result: <0.02%

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“Auto” PDA Wavelength Calibration Check

Anthracene

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Range / Linearity Test

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HPLC System Validation: Bringing it all together

VALIDATED
HPLC SYSTEM

PERFORMANCE METHOD
QUALIFICATION VALIDATION

CERTIFIED TRAINED
SYSTEM OPERATOR

SYSTEM
IQ /OQ

AUTOSAMPLER DETECTOR PUMP

IPV - IQ/OQ IPV - IQ/OQ IPV - IQ/OQ

PE SERVICE CERTIFIED
ENGINEER METROLOGY PERSON

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 Validation References
JR Jordan, “ISO 9000 and analytical instruments,” LC.GC Mag. 11(8), 1993.
”Good Laboratory Practice Regulations”, AF Hirsch (Ed), Marcel Dekker, NY, 1989.
FDA Compliance Guide for GLP, TN 92-42, 2/4/92.
21 Code of Federal Regulations Part 210:”cGMP in manufacturing, processing,
packing, or holding of drugs”, April, 1988.
Good Automated Laboratory Practices, EPA OIRM GALP publication draft, US
EPA, December, 1990.
LJ Goren, ”Computer system validation: Evaluating vendor software”, BioPharm,
February, 1989.
J Harris et al, “Validating concepts for computer systems used in the manufacture of
drug products”, Pharm Tech, May, 1986.
L Huber, Validation of computerized analytical systems, Interpharm Press, IL, 1995.
MW Dong et al, “A new Windows-based HPLC system”, Amer Lab, November,
1993.

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