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Dr Steve WoodLGC Limited

LGC Experience in Production and Characterisation of Pure Substances &

the Role of NMIs in this context

4005

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• Outline• Overview of LGC Limited• LGC Reference Materials Production• Accreditation Status• Pure Substance Reference Materials

• Purification• Characterisation• Inter comparisons• Value assignment

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LGC Limited - Locations

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Activities at LGC Teddington

62%

8%

30%

Analytical Services

Contract R&D

Quality Services

• Forensic Science

• Food Safety

• Pharmaceutical

• Lifescience• Reference Materials

• Proficiency Testing

• Training

400 staff on Teddington site

8 %

30 %62 %

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LGC’s UK National RolesGovernment Chemist

• Referee Analyst (20 Acts of Parliament)

• Adviser to UK government & industry on regulations and scientific input

National Measurement Institute

• Delivers world-class measurement science & technology and provides traceable and increasingly accurate standards of measurement for use in trade, industry, academia and government

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R&T Division

• NMS ChemBio (Chemical Measurements)• Development of methods & standards for improved

chemical analysis

• NMS ChemBio (Biochemical Measurements)

• Reliable measurements for the biotechnology industry

• MET (Measurements for Emerging Technologies)• Tissue Engineering, Bioprocessing, Point of Care

Testing

• Programmes Supporting NMI Work

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RM Production Resources

• 5 staff experienced in RM production• 3 staff in calibration analysis

• Pure materials & alcohols• Technical support

• 20+ staff in mass spectrometry and separation science

• Statistics support• Quality support

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LGC Certified Reference Materials

• Matrix materials

• Food• Environment• Clinical• Industry• Forensic

• Single substance organic materials• Purity• Melting point• Enthalpy of fusion• Elemental content

~150 materialsPure RM production for > 20 years

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RM Production Planning

• Production Steps • Material specification• Sourcing, preparation and sub-

division• Homogeneity assessment• Stability assessment• Characterisation of the assigned

value(s)• Calculation of the assigned value

and its uncertainty• Documentation and storage of the

material• Sales and storage and distribution

• LGC Standards

Produce a written project plan for production of each reference material

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Relevant Guidelines• ISO/IEC 17025

• General Requirements for the Competence of Calibration and Testing Laboratories

• ISO Guide 34• General Requirements for

the Competence of Reference Material Producers

• ISO Guide to the Expression of Uncertainty in Measurement (GUM)

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Material Certification

• Certification Panel• In house group• Project team members• Independent experts (e.g. statistics)• Quality Team representative

• European Reference Materials Co-operation• Technical review by experts from German (BAM)

and European (IRMM) metrology and reference material organisations

• ERM Panel approval

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Activities of Certification Panel• Review the intended use/original specification of the material• Review the expertise of the RM producer / subcontractors• Review the approaches used to characterise the property values• Examine records on the sourcing and preparation of the RM• Review the approaches used to assess homogeneity and

stability• Review supplementary information from the characterisation

studies• Review statistical processing of raw analytical data and results• Record the essential details of the review and examination of

data• Approve documentation for the reference material• Officially authorise the release of the RM for use

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Certificate (ISO Guide 31)

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International Inter-comparison StudiesPu

rity-

P20b

[LG

C]

Purit

y-P2

0c1

[LG

C]

Purit

y-P2

0c2

[LG

C]

Purit

y-P2

0d [L

GC

]

Purit

y-P2

0e1

[LG

C]

Purit

y-P2

0e2

[LG

C]

-4-2

02

4Z-

Sco

res

• LGC has demonstrated its competence in the measurement of the purity of organic substances through participation in the BIPM CCQM P20 series of inter-comparison studies on the following materials:• CCQM-P20.b: Ortho-xylene• CCQM-P20.c: Atrazine (2

samples, high and low purity)• CCQM-P20.d: Chlorpyrifos• CCQM-P20.e: Theophylline (2

samples, high and low purity)

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Comparison with other NMIs

Type scale: Total Location: MMmedianDispersion: sMMshorth

3210-1-2-3

LGCCENAMNMiANMIJNISTNIM

Concordance

App

aren

t Pre

cisi

on

• LGC’s performance in these studies is compared with the national measurement institutes from Australia (NMIA), China (NIM), Japan (NMIJ), Mexico (CENAM) and the USA (NIST)

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Brazil

BIPM

LAB A LAB B

UK

LAB DLAB C

INTERNATIONAL

NATIONAL

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Production of pure substance RMs at LGC• > 20 years• Accredited in 2006 by UKAS

for RM production to ISO Guide 34 & ISO/IEC 17025 • wide accreditation scope• including pure organic

compounds.• Pure organic certified RMs at

LGC:• Melting point materials• Malachite Green / LMG• Solvent Yellow 124• Kavain• Theophylline• Digoxin

www.ukas.com – for accreditation schedule

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Multiple Independent Methods• Used for pure materials

• e.g. Theophylline, kavain, saccharin, BHA, BHT• multiple methods

• HPLC• GC-FID / GC-MS• DSC

• calculate a mean value• correct for moisture and ash content • determine uncertainty

• Moisture • Karl Fischer titration• loss on heating

• Ash content • ICP-OES/MS• Ashing• Thermal Gravimetric Analysis

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Purification by chromatography

* 60Å, Particle size 40-63 micron, Fisher, Davisil

Silica gel (300 g)

Sintered glass

Ethyl acetate

MG + impurities(5 g)

Sintered funnel (Sintered funnel ( =100 mm)

Silica gel (300 g)

Sintered glass

Ethyl acetate

MG + impurities(5 g)

Sintered funnel (Sintered funnel ( =100 mm)

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Silica gel

Sintered glass

Ethyl acetate

MG

impurities Silica gel

Sintered glass

Ethyl acetate

MG

impurities

Purification by chromatography

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Silica gel

Sintered glass

Ethyl acetate

Silica gel

Sintered glass

Ethyl acetate

Purification by chromatography

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Silica gel

Sintered glass

Ethyl acetate

Silica gel

Sintered glass

Ethyl acetate

Purification by chromatography

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Silica gel

Sintered glass

Ethyl acetate

Silica gel

Sintered glass

Ethyl acetate

Purification by chromatography

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Silica gel

Sintered glass

MethanolSilica gel

Sintered glass

Methanol

Purification by chromatography

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Silica gel

Sintered glass

MethanolSilica gel

Sintered glass

Methanol

Purification by chromatography

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Silica gel

Sintered glass

MethanolSilica gel

Sintered glass

Methanol

Purification by chromatography

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Silica gel

Sintered glass

MethanolSilica gel

Sintered glass

Methanol

Purification by chromatography

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Silica gel

Sintered glass

Methanol

Evaporation

Silica gel

Sintered glass

Methanol

Evaporation

Purification by chromatography

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SFE vessel

Heater/Chiller

Collecting vessel

Pressureregulator

CO2 Pump

sCO2 Pump

Purification by Supercritical Fluid Extraction

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CollectionVessel

SFEVessel

Supercritical CO2

Pump

Gas

Expansion valve

Supercritical Fluid Extraction - Principle

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CollectionVessel

SFEVessel

Supercritical CO2

Pump

Gas

Expansion valve

Supercritical Fluid Extraction - Principle

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CollectionVessel

SFEVessel

Supercritical CO2

Pump

Gas

Expansion valve

Supercritical Fluid Extraction - Principle

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CollectionVessel

SFEVessel

Supercritical CO2

Pump

Gas

Expansion valve

Supercritical Fluid Extraction - Principle

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Case Study 1 Case Study 1 –– TheophyllineTheophylline

N

N

N

ONH

O

H3C

CH3

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Theophylline• Commercial supply:

• Confirmation of identity by mass spectrometry and 1H NMR. • Packaged into appropriate numbered glass vials (septum

and crimp capped). • Tested for homogeneity.

• Characterization using as many quantitative techniques as possible, in this case:• DSC,• HPLC UV, • GC-FID.

• Moisture determined using oven Karl Fischer. • Inorganic screen by ICP-OES & ICP-MS • Impurity identities confirmed by LC-MS

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HPLC-DAD

3.90

4

11.8

71

22.0

90

AU

-0.0010

-0.0008

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

0.0022

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peak

Impurity 1

Theophylline Impurity 2

3.90

4

11.8

71

22.0

90

AU

-0.0010

-0.0008

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

0.0022

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peak

Impurity 1

Theophylline Impurity 2

1.32

0

AU

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peakIm

purity 1

Theophylline

Impurity 2

Theobromine

Caffeine

1.32

0

AU

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peakIm

purity 1

Theophylline

Impurity 2

Theobromine

Caffeine

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11.930 Extracted

272.2

377.9

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

nm300.00 400.00 500.00 600.00 700.00

Spectral Properties of Theophylline

4.140 Extracted

272.2

354.2374.3 488.9516.8 642.3668.0698.6

AU

0.000

0.002

0.004

0.006

nm300.00 400.00 500.00 600.00 700.00

Spectral Properties of Impurity 1

23.993 Extracted

273.4

354.2377.9469.5488.9535.0 639.9668.0710.8AU

0.000

0.002

0.004

0.006

0.008

nm300.00 400.00 500.00 600.00 700.00

Spectral Properties of Impurity 2

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Certified Value• HPLC (n=9, s.d = 0.004) 99.938 %• DSC (n=6, s.d. = 0.01) 99.98 %

Unweighted mean used to assign the purity value for this material

• Impurity 1: 0.02 %• Impurity 2: 0.05 %• KF moisture (n=2, s.d. = 0.0071) 0.015 %• Inorganics (ICP-OES / ICP-MS) < 0.1 %

• Overall Purity: 99.95 %• Standard uncertainty (uchar) 0.022 %

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Stability• Accelerated testing

• e.g. for temperature

• Carried out under isochronous conditions• Removes long term analytical variations due to

• instrument drift• use of different calibration materials• changing environmental conditions• changes in analyst or laboratory reagents etc

• Long term testing• Compare results with samples stored under

‘reference’ conditions

Storage Temperature Storage Time (months) 40°C 20°C 4°C -18°C

1 a a a a 2 a a a a 3 a a a a

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Expiry Date & Uncertainty

• Stability testing• Expiry date - when a material is considered

no longer suitable for sale• Stability uncertainty (ustab)

• Uncertainty22

hom2

stabcharCRM uuukU ++×=

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Theophylline• Commercial supply:

• Confirmation of identity by mass spectrometry and 1H NMR. • Packaged into appropriate numbered glass vials (septum

and crimp capped). • Tested for homogeneity.

• Characterization using as many quantitative techniques as possible, in this case:• DSC,• HPLC UV, • GC-FID.

• Moisture determined using oven Karl Fischer. • Inorganic screen by ICP-OES & ICP-MS • Impurity identities confirmed by LC-MS

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HPLC-DAD

3.90

4

11.8

71

22.0

90

AU

-0.0010

-0.0008

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

0.0022

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peak

Impurity 1

Theophylline Impurity 2

3.90

4

11.8

71

22.0

90

AU

-0.0010

-0.0008

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

0.0022

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peak

Impurity 1

Theophylline Impurity 2

1.32

0

AU

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peakIm

purity 1

Theophylline

Impurity 2

Theobromine

Caffeine

1.32

0

AU

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Injection peakIm

purity 1

Theophylline

Impurity 2

Theobromine

Caffeine

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11.930 Extracted

272.2

377.9

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

nm300.00 400.00 500.00 600.00 700.00

Spectral Properties of Theophylline

4.140 Extracted

272.2

354.2374.3 488.9516.8 642.3668.0698.6

AU

0.000

0.002

0.004

0.006

nm300.00 400.00 500.00 600.00 700.00

Spectral Properties of Impurity 1

23.993 Extracted

273.4

354.2377.9469.5488.9535.0 639.9668.0710.8AU

0.000

0.002

0.004

0.006

0.008

nm300.00 400.00 500.00 600.00 700.00

Spectral Properties of Impurity 2

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Certified Value• HPLC (n=9, s.d = 0.004) 99.938 %• DSC (n=6, s.d. = 0.01) 99.98 %

Unweighted mean used to assign the purity value for this material

• Impurity 1: 0.02 %• Impurity 2: 0.05 %• KF moisture (n=2, s.d. = 0.0071) 0.015 %• Inorganics (ICP-OES / ICP-MS) < 0.1 %

• Overall Purity: 99.95 %• Standard uncertainty (uchar) 0.022 %

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Stability• Accelerated testing

• e.g. for temperature

• Carried out under isochronous conditions• Removes long term analytical variations due to

• instrument drift• use of different calibration materials• changing environmental conditions• changes in analyst or laboratory reagents etc

• Long term testing• Compare results with samples stored under

‘reference’ conditions

Storage Temperature Storage Time (months) 40°C 20°C 4°C -18°C

1 a a a a 2 a a a a 3 a a a a

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Expiry Date & Uncertainty

• Stability testing• Expiry date - when a material is considered

no longer suitable for sale• Stability uncertainty (ustab)

• Uncertainty22

hom2

stabcharCRM uuukU ++×=

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Case Study 2 Case Study 2 –– KavainKavain

O O

O

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Kavain characterisation

99.5

99.6

99.7

99.8

99.9

100

DSC HPLC GC-FID

Raw dataCorrected

Purit

y (%

)

Method

Error bars show 95% confidence interval

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Kavain: Uncertainty contributions

0.000 0.020 0.040 0.060 0.080 0.100

Puritydetermination

Moisture correction

(In)homogeneity

(In)stability

Total

Standard uncertainty (% purity)

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Issues & challenges

• Thermally unstable materials• Not amenable to thermal techniques e.g. gas

chromatography or differential scanning chromatography

• Materials with no chromophore• E.g. some amino acids

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Further information

• Recent/pending publications• Production of malachite green oxalate and

leucomalachite green reference materials certified for purity. Anal Bioanal Chem. Published on line 16 April 2008

• Development and accreditation to ISO/IEC 17025 calibration status of a melting point measurement facility for the UK. thermochimica acta (submitted)

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Acknowledgements

• Thierry le Goff• Ben Joseph

• UK Department of Innovation, Universities and Skills • Chemical and Biological Metrology Knowledge

Base Programme• www.nmschembio.org.uk