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ICP-MS in pharmaceutical analysis November 2007

Analysis of inorganic impurities in pharmaceuticals by ICP-MS

What are the applications and what are the benefits? Life Sciences Solutions Unit

Katja Kornetzky Solutions Manager for Process Development & Manufacturing QA/QC November 2007

ICP-MS in pharmaceutical analysis November 2007

Overview

• ICP-MS: The Technique
• Applications in Pharmaceutical Analysis
• The Agilent ICP-MS Compliance Software
• Conclusion
• Literature

ICP-MS in pharmaceutical analysis November 2007

What is ICP-MS?

Inductively Coupled Plasma Mass Spectrometry

Elemental analysis with:

• Wide elemental coverage
• Very low detection limits
• Fast analysis times (all elements at once)
• Wide analytical working range (up to 9 orders)
• Simple spectra
• High matrix tolerance
• Isotopic information

Viable alternative to ICP-OES or GFAAS*

*OES = Optical Emission Spectrometry, GFAAS = Graphite Furnace Atomic Absorption Spectrometry

ICP-MS in pharmaceutical analysis November 2007

Agilent 7500cx ICP-MS System with Collision Reaction Cell (CRC)

Low flow sample introduction system High temperature 27MHz plasma generator Multi-element interference removal by

• n-axis octopole reaction cell

High frequency hyperbolic quadrupole Fast simultaneous dual mode detector (9 orders dynamic range) Off-axis lens Cell gas inlet Octopole Plasma

ICP-MS in pharmaceutical analysis November 2007

Why is ICP-MS unique?

ICP-MS can:

• measure almost any element at ppt to ppm levels in almost any material
• measure all elements in a single analysis
• distinguish different element species (speciation)

Main requirements in pharmaceutical analysis are:

• high sensitivity
• good matrix tolerance
• low levels of interferences
• ease of coupling to speciation techniques (CE, IC, LC and maybe GC)

ICP-MS in pharmaceutical analysis November 2007

Pharmaceutical applications

Pharmaceutical waste water (EPA-regulated) Drug discovery / drug development:

• Analysis of individual forms of drug compounds using target element analysis
• Simple metal analysis during development of metal-based drugs

QA/QC and process development:

• National Pharmacopeia (e.g. USP, EP, JP) Testing

– Impurity limit tests – Metals in active pharmaceutical ingredients (API)

• QC of natural products – toxic impurities
• Toxic element impurities (e.g. heavy metals)

Clinical trials:

– Simple metal analysis for active component confirmation – Monitoring of the metabolites of an administered drug

ICP-MS in pharmaceutical analysis November 2007

Regulated pharmaceutical and clinical applications

Pharmacopeia Testing

• Impurity limit tests (general tests or based on individual monographs)*
• Quantitative metal content of API (USP monographs), e.g. Pt in platinum-containing drugs*

QC of natural products

• Heavy metal impurities in herbal remedies
• Heavy metal contaminations in TCM** (ICP-MS in China Pharmacopeia since June 1, 2005)

Metal Impurities

• Catalyst or reagent residues in raw materials, pharmaceutical substances and final products

as well as packaging materials (extractables/leachables)

• New EMEA draft guideline: specification limits for metal catalysts residues

Clinical Trials

• Metallodrugs (e.g. Pt-containing drugs for cancer treatment)

* None of these use ICP-MS. Instead Atomic Absorption Spectrophotometry (AAS), colorimetry and gravimetry are applied.

** TCM = Traditional Chinese Medicines

ICP-MS in pharmaceutical analysis November 2007

Conventional ICP-MS applications

Simple metal analysis in pharmaceuticals, raw materials and clinical samples

ICP-MS in pharmaceutical analysis November 2007

USP Methodology

• Atomic Absorption Spectroscopy

– e.g. USP limit tests for <206> Al, Cr*, Rh*, Mo**

• Colorimetry

– e.g. USP limit tests for <231> heavy metals, <241> Fe, <251> Pb, <211> As

• Titration

– e.g. USP limit test for <261> Hg

• UV spectroscopy

– e.g. USP limit tests for <291> Se, <211> As

• Gravimetry

– e.g. Pt content in Cisplatin, Carboplatin (UPS monographs)

*Alprostadil monograph ** Monograph for ammonium molybdat injection

ICP-MS in pharmaceutical analysis November 2007

QC of traditional Chinese medicines (TCMs)

Chinese Pharmacopoeia 2005, Appendix IX

Agilent Application Notes:

• Determination of Toxic Elements in Traditional Chinese Medicine Using Inductively

Coupled Plasma Mass Spectrometry, P/N 5989-5591EN

• Evaluation of Conventional ICP-MS and ORS-ICP-MS for Analysis of Traditional

Chinese Medicines, P/N 5989-2570EN

NEW !

ICP-MS in pharmaceutical analysis November 2007

Example: Determination of heavy metals in herbal medicines

Shugan pill exceeds legal limits for Hg (0.2 ppm) and As (2.0 ppm)

[2005 Pharmacopoeia of the People’s Republic of China]

Agilent Application Note 5989-5591EN

We wish to thank Hua Zhang, Yan-zhi Shi and Ying-feng Wang at the Capital Normal University in Beijing, China for their permission to publish the results from their scientific study*

*Hua Zhang is a Postgraduate Student in the Chemistry Department and Yan-zhi Shi and Ying-feng Wang are Professors in the Analytical Center, all at Capital Normal University in Beijing, China.

ICP-MS in pharmaceutical analysis November 2007

Metal impurities – Leachables from pharmaceutical packaging materials

1. Interaction between formulation and packaging material results in components migrating into the drug product 2. These components may be toxic or affect the stability of the drug product 3. Storage conditions impact leaching (heat, UV radiation, storage time)

Typical leachables are:

• Small organic molecules [monomers, excipients, reaction by-products],
• Metal ions and trace elements (e.g. aluminum, cadmium, chromium, copper, lead,

manganese, and zinc)

“Metal ions can affect the stability of the formulation, catalyze the degradation

• f the active pharmaceutical ingredient (API) and cause unqualified

degradates to form, or pose a toxicity threat on their own.”

KYLE A. FLlSZAR, DAVID WALKER and LEONARDO ALLAIN, Profiling of Metal Ions Leached from Pharmaceutical Packaging Materials, PDA Journal of Pharmaceutical Science and Technology, Vol. 60, No.6, November-December 2006

ICP-MS in pharmaceutical analysis November 2007

Guideline on the specification limits for residues of metal catalysts

EMEA* draft guideline dated January 2007

Objective:

“Recommend maximum acceptable metal residues arising from the use of metals as catalysts or reagents in the synthesis of drug substances and excipients.” Since the use of catalysts or reagents is restricted to defined chemical reactions in the synthesis of pharmaceutical substances, limitation of residues in these substances is sufficient, and in general there is no need to set limits for metal residues in the final medicinal products containing these substances.

* European MEdicinal Agency: Its main responsibility is the protection and promotion of public and animal health, through the evaluation and supervision of medicines for human and veterinary use. The EMEA is responsible for the scientific evaluation of applications for European marketing authorization for medicinal products

ICP-MS in pharmaceutical analysis November 2007

Metal catalyst residues

EMEA classification and concentration limits

ICP-MS in pharmaceutical analysis November 2007

ICP-MS for routine analysis in drug discovery & clinical trials

Clinical samples require

• High sensitivity (measurement of low-level trace elements)
• Excellent matrix tolerance (biological matrices)
• Large number of samples analyzed daily

Matrix tolerance is an essential part of clinical analysis

• High potential for matrix contamination of the ICP-MS system, if the

matrix is not effectively decomposed

• Resulting deposits may adversely affect instrument performance
• Efficiency of plasma determines matrix tolerance

ICP-MS in pharmaceutical analysis November 2007

Example 1: sanofi-aventis (UK)

High throughput assay for oxaliplatin

Success Story

• Pub. No.

5989-7077EN Increased sample throughput and improved reliability with an Agilent 7500a ICP-MS

“An Agilent 7500 ICP-MS instrument was chosen as the replacement equipment because of the excellent matrix tolerance, lower backgrounds, larger dynamic range and smaller footprint.”

ICP-MS in pharmaceutical analysis November 2007

Example 2: Long-term sample analysis

Matrix tolerance is of major importance

An efficient plasma design:

• reduces matrix load on the interface

and vacuum system (less signal drift)

• reduce the maintenance
• increases ionisation efficiency of key

trace elements, e.g. Zn, Cd and Hg

• removes interferences by efficient

decomposition of matrix-based polyatomic species

Isla Strang and Alan Cox with 2 Agilent 4500 ICP-MS systems. Courtesy of Centre for Analytical Sciences, Sheffield

ICP-MS in pharmaceutical analysis November 2007

Advanced ICP-MS applications – speciation

ICP-MS as a sensitive detector with high specificity for established separation techniques

ICP-MS in pharmaceutical analysis November 2007

What is speciation?

*J. A. Caruso, K. L. Sutton, and K. L. Ackley, ”Elemental Speciation - New Approaches for Trace Element Analysis,”

• Chap. 1, p. 1, Volume XXXIII of COMPREHENSIVE ANALYTICAL CHEMISTRY. Elsevier Publishing, Netherlands, 2000.

“Elemental Speciation - the analyses that lead to determining the distribution of an element’s particular chemical species in a

• sample. This may be further associated with
• xidation state, organometallic nature or

complex form.”*

ICP-MS in pharmaceutical analysis November 2007

Why do we need speciation?

Almost all elements form species which can alter their toxicity and mobility

• CrIII vs CrVI; inorganic As vs organic As; humic acid complexes …

Some species vary in toxicity or therapeutic activity

• Pt-containing drugs in cancer treatment

– Anti-tumour activity versus side effects of drugs and metabolites – Species with different structure and Pt oxidation state

• Medical research on capability of proteins in binding different (toxic) metals

– Metallothionein isoforms

• Heavy metal or pesticide impurities in natural products

– For example, organomercury or organophosphorus species

• Speciation of heteroatom-containing drugs (e.g. Cl, Br, S, P)

Need simple, selective, rapid, sensitive and accurate determination

• f these species

ICP-MS in pharmaceutical analysis November 2007

Practical coupled techniques for ICP-MS

ICP-MS is suitable for coupling to many separation methods:

• Capillary Electrophoresis (CE)
• Ion Chromatography (IC)
• Liquid Chromatography (HPLC)
• Gas Chromatography (GC)

ICP-MS in pharmaceutical analysis November 2007

Suitability of ICP-MS for chromatography

• Suitable for liquid or gas phase
• Usable with a wide range of flow rates

– Liquid phase: less than 20µL/min (capillary LC) to 2mL/min (standard LC)

• Less than 1uL/min flow (e.g. nano-LC or CE) can be handled, with a make-up solution

– Gas phase: Conventional GC carrier gas flow rates can be used, with a make-up Ar flow (~1L/min)

• Tolerates a variety of LC mobile phases

– Organic solvents, gradients from aqueous to organic – High salt buffers

• High sensitivity for most elements
• Rapid data acquisition

– sufficient # of data points across chromatographic peak

• Ability to measure isotopes (isotope tracer studies, isotope dilution calibration)
• Mass selective and extremely specific

– mass spectrum almost free from inter-element effects – isotopes of all elements are free from direct

• verlap from another element

ICP-MS in pharmaceutical analysis November 2007

CE interface for ICP-MS

Photo courtesy of Dr Andreas Prange, GKSS, Germany

Instrumentation:

• Agilent 3D CE
• Cetac CEI-100 interface
• Agilent 7500s ICP-MS

ICP-MS in pharmaceutical analysis November 2007

CE interface for ICP-MS

CE interface requires

• make-up liquid flow to carry eluent to ICP-MS nebulizer
• very low flow nebulizer and low-volume spray chamber

for efficient aerosol transport and preservation of chromatographic separation

Photo courtesy of Dr Andreas Prange, GKSS, Germany

ICP-MS in pharmaceutical analysis November 2007

Speciation in medical research

CE-ICP-MS for separation of metallothioneins (MT)

Metallothioneins are

• A group of proteins
• Capable of binding physiological and

xenobiotic heavy metals – CE preserves the molecular structure

• f high molecular weight (MW)

compounds – speciation of high MW molecules, such as metallothionein isoforms

Courtesy of Dr Andreas Prange, GKSS, Germany

CE-ICP-MS is suitable for the rapid speciation

• f elemental species

ICP-MS in pharmaceutical analysis November 2007

Speciation in medical research

CE-ICP-MS analysis of MT in brain tissue

Courtesy of Dr Andreas Prange, GKSS, Germany

ICP-MS in pharmaceutical analysis November 2007

LC-ICP-MS – first hyphenated technique to be seriously investigated

nebulizer & spray chamber gas controller ICP torch turbo molecular pump Q-pole mass filter Ar gas rotary pump turbo molecular pump liquid chromatograph

The Agilent 1100 LC coupling provides

• Integrated sequencing for routine analysis
• Use of organic mobile phases

Agilent 1100 LC coupled directly or via split to 7500 ICP-MS

ICP-MS in pharmaceutical analysis November 2007

11.2011.4011.6011.8012.0012.2012.4012.6012.8013.0013.2013.4013.60 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 160000 170000 180000 Time--> Abundance Ion 79.00 (78.70 to 79.70): 000_STD.D Ion 79.00 (78.70 to 79.70): 002_STD.D Ion 79.00 (78.70 to 79.70): 003_STD.D Ion 79.00 (78.70 to 79.70): 004SMPL.D Ion 79.00 (78.70 to 79.70): 005SMPL.D Ion 79.00 (78.70 to 79.70): 006SMPL.D Ion 79.00 (78.70 to 79.70): 007SMPL.D

Standards: 0.0, 0.02, 0.05, 0.1mg Samples (3 replicates):

• 1.0 mg of Cl compound 1
• 0.1 mg of all other

compounds

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 160000 170000 180000 190000 Time--> Abundance Ion 35.00 (34.70 to 35.70): 000_STD.D Ion 35.00 (34.70 to 35.70): 002_STD.D Ion 35.00 (34.70 to 35.70): 003_STD.D Ion 35.00 (34.70 to 35.70): 004SMPL.D Ion 35.00 (34.70 to 35.70): 005SMPL.D Ion 35.00 (34.70 to 35.70): 006SMPL.D Ion 35.00 (34.70 to 35.70): 007SMPL.D

Data courtesy of Martin Gray, Sanofi-Synthelabo

Speciation for pharmaceutical samples

Determination of Cl, Br & S heteroatoms

Separation and quantification

• f 3 Cl compounds (left)

and 1 Br compound (right) in a mixed sample

35Cl 79Br

ICP-MS in pharmaceutical analysis November 2007

Speciation in clinical research

Pt compounds in anti-tumor drugs

Data courtesy of Peter Galettis, University of Auckland, New Zealand

ICP-MS in pharmaceutical analysis November 2007

Speciation in clinical research

Pt compounds in anti-tumor drugs

What is Cisplatin?

• An anti-neoplastic drug with proven effectiveness against cancer
• One of the most important anti-tumor drugs in clinical oncology

But there are some side effects:

• For example nephrotoxicity and nausea,
• Some result from the activity of metabolite products from the administered drug

As a result:

• search for second generation platinum compounds with less nephrotoxicity and greater anti-

tumor activity.

• To investigate the behavior of platinum compounds, a selective and sensitive analytical

method is required.

LC-ICP-MS is ideal for this application.

Peter Galettis, University of Auckland, New Zealand

ICP-MS in pharmaceutical analysis November 2007

Speciation in clinical research

Example: Pt species in compound JM216

Data courtesy of Peter Galettis, University of Auckland, New Zealand

Chromatogram for 2.29 mg/L of the main compound JM216 [Bis (acetato) amine-dichloro (cyclohexylamine) platinum (IV)], Agilent application P/N 5968-8185E

The combination of HPLC with ICP-MS detection enables the determination of Pt compound impurities down to ng levels.

JM216 JM216 JM216

ICP-MS provides the sensitivity and specificity to analyze trace level of Platinum compounds with highest throughput.

ICP-MS in pharmaceutical analysis November 2007

Advanced ICP-MS applications – speciation

Combined elemental and organic spectrometry 1) ICP-MS combined with UV detection (conventional LC detector with flow-through cell) 2) ICP-MS combined with molecular mass spectrometry to give elemental detection and quantification, as well as molecular structure and compound identification

ICP-MS in pharmaceutical analysis November 2007

Laser Ablation ICP-MS

Analysis of solid sample surfaces

Merchantek UP 213 Laser Ablation System linked to Agilent 7500 ICP-MS

ICP-MS in pharmaceutical analysis November 2007

Principles of laser ablation (LA) ICP-MS

LASER - Light Amplification by Stimulated Emission of Radiation Procedure:

• Laser beam focused onto sample surface in ablation chamber or cell
• Miniature plasma above sample ablates material from surface
• Resultant particulate material is transported to ICP-MS with carrier gas stream (e.g. Ar)
• Sample is decomposed, atomised and ionized in ICP plasma and analyzed in mass

spectrometer

Applications for LA-ICP-MS:

• Monitor distribution of administered drugs among different tissues and body compartments
• In-situ analysis of metals and other elements in samples separated using PAGE*, e.g.:

– Au and Pt drug metabolite identification – Determine degree of phosphorylation of different separated proteins

* Polyacrylamide gel electrophoresis

ICP-MS in pharmaceutical analysis November 2007

In-situ direct tissue analysis

Example: Analysis of brain tissue (rat)

Brain tissue – the laser sampling pattern can be seen on the rat brain tissue sample

Courtesy of Centre for Analytical Sciences, Sheffield

After administration of the drug, the test animal is freeze-dried, encased in polymer and

• microtomed. Individual sections are then analyzed using laser ablation.

ICP-MS in pharmaceutical analysis November 2007

Example: Element distribution in rat brain sample after drug treatment

Distribution of different elements in tissue sections

Elements shown are Mg, Fe and P

Courtesy of Centre for Analytical Sciences, Sheffield

31P 26Mg

Fe

ICP-MS in pharmaceutical analysis November 2007

Use of ICP-MS in regulated environments

Complying with 21 CFR Part 11

Agilent’s solution for ICP-MS ensures…

… data security by:

• Secure central storage of data
• User access limited to authorized individuals

… data integrity by:

• Automatic data storage and versioning
• Full version control for ALL relevant data including original result reports
• Integrated archival and long-term storage in a content management system

… data traceability by:

• Automatic user-independent audit trail
• Electronic signatures

Full support of all requirements mandated by 21 CFR Part 11 in closed system

ICP-MS in pharmaceutical analysis November 2007

Complying with 21 CFR Part 11

More details

“Agilent ICP-MS ChemStation – Complying with 21 CFR Part 11”

Agilent Application Note Publication number: 5989-4850EN

http://www.chem.agilent.com/scripts/Litera turePDF.asp?iWHID=45817

ICP-MS in pharmaceutical analysis November 2007

Use of ICP-MS in regulated environments

Boosting productivity

Agilent’s solution for ICP-MS adds value by…

…fully supporting your workflow with:

• Data review independent of ICP-MS ChemStation
• Extensive search and retrieve capabilities
• Data access from anywhere using a web browser
• One central repository for all your data and related documents

(e.g. SOP documents, electronic reports, spreadsheets, …)

… and enabling easy administration with:

• Standard Windows user administration
• Scalable solution – from single instrument to enterprise system
• Integrated data archival

ICP-MS in pharmaceutical analysis November 2007

Literature

• High Throughput Assay for Oxaliplation: P/N 5989-7077EN
• Determination of Toxic Elements in Traditional Chinese Medicine

Using Inductively Coupled Plasma Mass Spectrometry, P/N 5989- 5591EN

• Evaluation of Conventional ICP-MS and ORS-ICP-MS for Analysis of

Traditional Chinese Medicines, P/N 5989-2570EN

• Agilent ICP-MS Journal # 32: Including article on compliant ICP-MS
• Handbook of Hyphenated ICP-MS Techniques