identification of unknowns in lc-ms using a holistic approach · • fragmentation pattern checked...

Identification of Unknowns in LC-MS Using a Holistic Approach

From material knowledge to confirmed hits

Dr. Roberto MenzelSartorius Stedim Biotech GmbH

6th European Forum on Extractables, Leachables, Medical Devices and Food Contact Materials Testing

Agenda

Short Introduction into Sartorius

Extractables and Leachables

Screening in LC-HRMS measurements

Chemistry of Extractables

Case Study

25th Sept. 2019 Page 4

Global player with around 60 sales and production sites

India, Thailand, Singapore, Vietnam,China, Malaysia, South Korea, Japan,Australia

Netherlands, Belgium, Ireland, Israel,UK, France, Spain, Sweden, Finland, Denmark, Germany, Poland, Austria, Hungary, Switzerland, Italy, Russia, Tunisia, South Africa

EMEA

Am

eric

as

Asi

a |

Paci

ficUSA, Canada, Puerto Rico, Mexico, Brazil, Argentina

Production and Production/Sales

Sales


Main location in Göttingen, Germany


Roots of both divisions reach far back

Weighing TechnologyCore of Lab Division

Filtration Core of Bioprocess Division

University mechanician | Company founderFlorenz Sartorius invents short-beam analytical balance

Wilhelm Sartorius founds joint venture with Nobel laureate and creator of membrane filtration Richard Zsigmondy1

1870 1927

1) Zsigmondy, R. and Bachmann, W. “Über Neue Filter” Z. anorg. allg. Chem., 103(1), 119–28, (1918)


High initial cost

Considerable cleaning effort

Risk of cross-contamination

Pioneer in single-use manufacturing technologies

PresentPast

+ CAPEX reduction over entire lifecycle

+ Lower water and energy consumption

+ Higher flexibility


Well positioned as a total solution provider across the biopharma process chain

Fermentation Cell Harvesting Buffer | PreparationCell Culture Media

Process Development Production

Services

Purification


Extractables and Leachables – Do Not Get Confused!

The terms Extractables and Leachables are clearly defined

• Extractables = potential impact

• Leachables = actual impact

• Extractables = test the material SUS/component

• Leachables = test the final productSuppliers responsibility

Biopharma responsibilitySartorius supports customer viaCONFIDENCE® Validation Services

Extractables Guides

Leads to two objectives of testing scenarios

The impact of a single use product or container closure systems


Extractables – Regulatory Status for SU Equipment

• There are as yet no single specific standards or guidance for SU equipment for

Extractables (and Leachables) testing

• USP DRAF <665> and BPOG protocol (2014) provide practical information on “what,

when and how” to perform an Extractables Study (Time, Temp., Shaking, )

• Sartorius developed an Extractables Approach for material characterization and safety

assessment for single-use equipment 1

Design and Installation

Qualification

Operational Qualification

Performance Qualification

Implementing new equipment into a pharmaceutical process

Extractables data “Simulation” data Leachables data

Required to identify potential leachables

1) Pahl, I. et al. Development of a Standardized Extractables Approach for Single-Use Components - General Considerations and Practical Aspects. Bioprocess Int. 16, (2018).


Extractables & Sartorius

• Sartorius provides comprehensive Extractables information since early 1995 1

• Extractables data to support customers in pharmaceutical & biopharmaceutical

industry in the DQ / IQ phase

• Laboratory at Sartorius since 2015

• Defined and standardized internal Extractables testing

• Data provided as Extractables guides (EG)

• Since 2018 LC-HRMS screening

results included in EG

• Active in research 2-5

1 Reif, O. W. et al. PDA J. Pharm. Sci. Technol. 50, 399–410 (1996)2 Dorey, S. et al. Ind. Eng. Chem. Res. 57, 7077–7089 (2018)3 Pahl, I. et al. Analysis and evaluation of single-use bag extractables for validation in biopharmaceutical applications PDA J. Pharm. Sci. Technol. 68, 456–71 (2014)4 Hauk, A., Jurkiewicz, E., Pahl, I., Loewe, T. & Menzel, R. Filtration membranes - Scavengers for leachables ? Eur. J. Pharm. Sci. 120, 191–198 (2018)5 Menzel, et al. Comparative Extractables Study of Autoclavable Polyethersulfone Filter Cartridges for Sterile Filtration. PDA J. Pharm. Sci. Technol. 72, 298-316 (2018)

Flexboy Extractables Guide 2014 Most current Extractables Guide 2019


Extractables & Sartorius – Equipment LC-HRMS

• Waters Xevo® G2-XS QTof with ACQUITY UPLC ready for use since Q1 2016

• ESI and APCI ionization available with LockSprayTM (mass accuracy below 1 ppm)

• Software is UNIFITM 1.8.2

PEG pattern obtained ESI pos using UNIFI software


Target, Suspect, and Non-Target Screening - Internal LC-MS Method

Sartorius method details in brief Rational

• Neutral eluent for detection of acidic and

basic compounds

• 1 µL Injection volume (sensitivity fine,

best chromatography, less ion

suppression)

• LC method extended up to 40 min for

unknown samples

• UV detection for weak ionizing

compounds

• Scan range up to m/z = 1500 including all

major additives

• Standard C18 column (10 cm)

Parameter Value ESI

Ionization polarity ESI positive and ESI negative

Mass range 50 - 1500 m/z

Cone voltage 40 V

Analyzer Mode Sensitivity

Capillary voltage ESI pos and neg: 3 kV

Source temperature 120 °C

Cone gas flow 50 L/h

Desolvation gas flow and temperature

950 L/min, 550 °C

LockSpray every 30 s

Ramp MSe Low CE: 4.00 eV, High CE: 40 eV

ConditionsFlow 0.5 mL/min, Column Temp. 40 °C, Injection Volume = 1 µL

Eluent A:10 mM NH4CH3COOH Water, B: Acetonitrile

Gradient

0 to 0.5 min B: 5%;0.5 to 9 min B 99%9 to 19.5 min B 99% (extended up to 40 min)19.6 to 20 min B 5%

UV/VisDetection @ λ = 200, 220, and 254 nm (and 3D λ = 220– 510 nm)

Column BEH C18 (1.7 µm, 2.1 x 100 mm)


Unknowns – Why Are They Such a “Nuisance”

• Evaluation of an Extractables profile includes a safety assessment

• All available toxicological information are used (PDE, NOAEL, LD50 values)

• The Threshold of Toxicological Concern (TTC) concept is applied for compounds with

known structure w/o toxicological data 1

• ICH M7 defined TTC of 1.5 µg/day (lifetime > 10 years) with negligible risk for

mutagenic compounds and typically applied also for Unknowns

• All structural additional information are beneficial, e.g. exclusion of groups

• Keep in mind – most E/L are of low toxicological concern 2

Quantity of a complete unknown compound might exceed allowable exposure because of (incorrect) lowest TTC – What then?

1) Koster, Sander et al. Application of the TTC Concept to Unknown Substances Found in Analysis of Foods. Food and Chemical Toxicology 49(8): 1643–60 (2011).

2) Li, K. et al. Creating a Holistic Extractables and Leachables (E&L) Program for Biotechnology Products. PDA J. Pharm. Sci. Technol. 69,590–619 (2015).


Target, Suspected-Target and Non-Target Screening in LC-HRMS

• Target Screening: Reference compounds available,

calibration, known validation parameters, etc.

→ quantitative data“Target-Screening”

“Unknown-Screening”

If possible, spent all you laboratory time to improve your suspected-target screening.

• Suspected-Target Screening: Expected analytes,

established database, known (relative) retention time,

ionization behaviour, response factors, fragmentation

pattern, etc.

→ quantitative and/or qualitative data

• Non-Target Screening: Low sample information, no

information chemical classes, etc.

→ qualitative data


Suspected-Target Screening – List of Suspects & Where to start?

• Suspected-target screening in LC-MS is extremely helpful in E/L studies

• The size of the list of suspect is not important – it’s the quality

• For plastic additives a start is Zweifels “Plastics Additives Handbook”

(Plastic additives are no secret – Manufacturer use these additives.) 1

• Evolve library suspects with measurement results

• Sartorius has about 150 secured suspects

and 300 additional additives implemented

Irganox® 1425 Maybe use this for suspect

Not blindly include structures

Tinuvin® 622EP/USP plastic additive

How to handle polymeric additives?

Maybe they are a good starting point

1) Most used antioxidants (Irgafos® 168, Irganox ® 1010, etc.): “Stabilizer systems of triaryl phosphites and phenols” DE 1976-2606358, 1976!


Non-Target Screening – Where to start?

• Non-Target Screening (NTS) is described in literature mainly for water or

biochemical analysis 1

• Possible workflow described in literature 2

• No existing guidelines on how to actually perform NTS in E/L studies

1 General Screening and MethodsKrauss, M., Singer, H. & Hollender, J. Anal. Bioanal. Chem. 397, 943–951 (2010)

Milman, B. L., Trends Anal. Chem. 69, 24–33 (2015)Schymanski, E. L. et al., Anal. Bioanal. Chem. 407, 6237–6255 (2015)

2 WorkflowLittle, J. L. et al., J. Am. Soc. Mass Spectrom. 22, 348–359 (2011)Little, J. L. et al., J. Am. Soc. Mass Spectrom. 23, 179–185 (2012)Müller, A.. et al., Chemosphere 85, 1211–1219 (2011)

• The best NTS or “Unknown” Screening is done by: a. Know your material, better know the additivesb. Think about and know your chemistryc. Read literature

Derive your possible structures and perform suspected-target screening

For confirmation authentic reference material should be used or very good justification.In chemistry, structure elucidation done by NMR, elemental composition, IR, etc.


Part 1

• General: Samples signal must exceed blank by 50%

• Visual comparison of the BPI of the sample vs reference blank

• Find unknown peak and corresponding mass signal

• Only the most intense monoisotopic peak is reported

• PDA (220 nm) is used to detect compounds not ionized well by ESI

• Elemental Composition tool is used to generate formula

• Obtained parameters e.g. used in Discovery Tool to perform structure elucidation

(ChemSpider)

• If no hit is obtained molecular formula is searched in Scifinder®

Non-Target Screening – Sartorius Approach

www.chemspider.com

www.cas.org


Non-Target Screening – Sartorius Approach

Part 2

• Does the structure make sense and are they reasonable for extraction solvent?

• Does the retention time make sense?

• Does the ionization behavior make sense?

If yes,

• Structure implemented in library, checking if peak is assigned by UNIFI

• Fragmentation pattern checked for reasonability

* United State Pharmacopoeia <1663> Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems. 38, 7166–7180 (2015).

RT MS

[min]

m/z

ESI pos

m/z

ESI neg

UV/Vis

signal

Molecular

Formulai-Fit Structural Suggestion (e. g. Name, CAS-#, origin)

Identification

Level

3.21 792.5992 850.6013 - C42H77N7O7 100 Caprolactam Heptamer (CAS 16056-00-1) confident

4.27 - 593.1297 - C30H26O13 100Ethylene glycol terephthalate (3:3) (CAS 16958-96-6),

suspect, PET degradantconfident

5.02 - 785.1715 - C38H22N14O7 35 Unknown unknown

6.63 594.1606 635.1406 medium C30H24O12 Ethylene terephthalate cyclic trimer (CAS 7441-32-9) confirmed

7.05 581.4317 579.4156 - C36H56N2O4 93 Irganox 1098 degradant (tert-Butyl split off) tentative

Example of a list of hits of a LC-MS suspect and non-target screening

Identification level should be given according to USP <1663>*


Chemistry of Extractables/Polymers – Simplify Your Screening

• Extractables are based and are formed from existing additives or processing aids or

from the polymeric materials

• Extractables can be traced back to the raw material

• Yes, also Extractables are subject to standard chemical principles

• Reactions with the extraction media are possible (e.g. Ethanol)

• Reactions between Extractables in an extraction solvent are unlikely (dilution)

• Analytical “False positive” hits are possible e.g. decarboxylation in GC-MS, wrong

blanks, bad analytics 1

General Statements

This means, all Extractables and potential “unknown” are explainable by material/manufacturing and chemical/analytical rationales.

1) Menzel, R. “Extractables Data from Single-Use Systems for the Biopharmaceutical Industry - Need Extractables be such a difficult and “controversial” topic?”, BPI West 2019, DOI: 10.13140/RG.2.2.15050.62403/1, https://tinyurl.com/y6c26fhn


• Esterification and hydrolysis,

transesterification

• Oxidation reaction

• Radical reactions

• Acetal formation

• Other nucleophilic substitution

reactions (very rare)

• Nitration/Nitrosation (very rare)

Total synthesis of (−)-colchicine

Remark: Catalyst (acids) produced during gamma irradiation

These are not the reactions mechanism one should expect for Extractables or

Leachables

Chemistry of Extractables/Polymers – Simplify Your Screening

Common Basic Chemical Reactions in E/L


Chemistry of Extractables - Examples Oxidation Reaction

Bolland, J L, and G. Gee. 1946. “Kinetic Studies in the Chemistry of Rubber and Related Materials. II. The Kinetics of Oxidation of Unconjugated Olefins.” Transactions of the Faraday Society 42(0), 236-43, 1946

„Bolland & Gee mechanism“ polymer autoxidation

Wanted oxidation Irganox® PS 800

Oxidation degradants of BHT

• Degradation of polyolefins is unavoidable

• Crosslinking and oxidation take place

Oxidation takes place also for the additives.


• Esterification, hydrolysis, or transesterification takes place in aqueous extraction

solutions but also during storage

• Process is acid-catalyzed

• Example: Irgafos® 168 - Secondary antioxidant polyolefins

Chemistry of Extractables/Polymers – Example Hydrolysis Ester

„Famous“ bDtBPP (CAS 69284-93-1)

Usually observed after irradiation

because of acid-catalyzed hydrolysis

Typically, not observed after long storage

Irgafos® 16831570-04-4

Irgafos® 168 oxidized95906-11-9


Chemistry of Extractables/Polymers – Example Acetal Hydrolysis

• Acetal formation - a reversible acid-catalyzed process

• Typically observed in aqueous extracts of gamma-irradiated test items

• Example: Irgaclear® DM - Clarifying agent for polypropylene 1

Irgaclear® DM(54686-97-4)

Sorbitol(50-70-4)Not detected by GC-MS or LC-MS

4-Methylbenzaldehyde(104-87-0)detected by GC-MS

1) McDonald, J. G. et al. “Identification and Quantitation of Sorbitol-Based Nuclear Clarifying Agents Extracted from Common Laboratory and Consumer Plasticware Made of Polypropylene.” Analytical Chemistry 80(14), 5532–41, 2008

Irgaclear® DM mono(w/o stereo 88449-62-1)(with stereo 126748-37)


Chemistry of Extractables/Polymers – Example Acetal Hydrolysis

Irgaclear® DMleft: ESI pos; right: ESI neg

Irgaclear® DM mono left: ESI pos; right: ESI neg

• Irgaclear® DM and its degradant „Irgaclear®

DM mono“ can be detected with LC-MS easily

• Compounds are present in aqueous extracts

XIC ESI pos, polypropylene extract


Case 1: Unknown Identification - Filter Cartridge

Material Information Filter CartridgeMembrane: PTFE

Support Fleece: Polypropylene 1

Core/cage: Polypropylene 2

End Caps: Polypropylene 2

Additive package partly known

Extraction Conditions

• Surface to volume ratio: 1:1; Effective Filter Area: 7500 cm2 Extraction volume:

7500 mL.

• Extraction media: WFI and ethanol

• Extraction parameters: t = 24 h, T = 40 °C, shaking speed = 75 rpm

Extractables study of a sterilizing-grade filter



LC-HRMS BPI Chromatogram ESI + ethanol extract (top sample, bottom blank chromatogram)

ESI positive

sample

blank

2Irgafos 168

Irgafos 168 (oxidized)

Irganox 3114

Unknown 1: 10.36 minUnknown 2: 11.13 min

1

Two intense signals in LC-HRMS chromatogram All other signals identified.



monoisotopic signal(M+H)

+

ESI pos fragment mass spectra high CE (40 eV) ESI pos fragment mass spectra high CE (40 eV)

ESI pos mass spectra low CE ESI pos mass spectra low CE

21

- 100 Da

- 40 Da

- 17 Da

- 100 Da

- 40 Da

- 17 Da

Unknown 2 is a homologue of Unknown 1 (+ C2H4) - longer alkane chainUnusual fragmentation pattern

monoisotopic signal(M+H)

+


Step 1: Find Reasonable Elemental Composition

• Monoisotopic mass ESI pos H+ adduct, reasonable elemental composition generated in

UNIFI Elucidation Toolset

→ Unknown 1: C27H53NO3 and Unknown 2: C29H57NO3

Step 2: Database Search - Discovery Tool

• Two possible “classes” of molecules for Unknown 1 identified

→ A) An hindered amino light stabilizer (HALS) or degradant thereof

→ B) Some detergent from a cleaning solution or added as flow improver

Candidate A (not available, some references) Candidate B (mainly patents)


“Representative” for Candidate A (available & known)


Step 3: Test Identified Candidates

• Purchase potential/similar candidate – confirmation or additional information

(fragmentation, ionization behavior)

→ “Rep.” Candidate A similar mass signal, different retention time, no trace of

unknown peaks

Step 4: Identify Source of the Extractables

• Extraction of the different raw materials

→ Extract of one polypropylene (PP1) showed the same unknowns

Step 5: Identification of Additives of the Raw Material

• Stearic acid, Palmitic acid, Irgafos® 168, and degradant from Tinuvin® 622

detected

→ On request, Tinuvin® 622 was confirmed as additive from supplier



Step 6: Develop Possible Alternatives

• Plausible structure from reactions of present additives

• Reaction product (transesterification) of a fatty acid (detected) and Tinuvin® 622

(confirmed) – This would be a very rare case!

• Identification of potential main reaction product – chemistry of esters

Reaction between both compounds takes place most likely during injection molding process (high temperature, trace water always present) and not during extraction.

Tinuvin® 622(65447-77-0, EP plastic additive 22, light stabilizer)

Palmitic acid - or salts thereof(57-10-3, releasing agent, ubiquitous)



1 2

Unknown 2: C29H57NO3

m/z 468.4411, RT 11.31 min confirmed

1-Hydroxy-2,2,6,6-tetramethyl-4-piperidinyl stearate (HTPES)No CAS available, not described in literature

Unknown 1: C27H53NO3

m/z 440.4098, RT 10.35 confirmed1-Hydroxy-2,2,6,6-tetramethyl-4-piperidinyl palmitate (HTPEP)No CAS available, not described in literature

from Tinuvin® 622 Palmitic acid Tinuvin 622 Stearic acid

Step 7: “Synthesis” – Feasibility study

• Difficult to purify, NMR showed product, purity ⋲ 70% (sufficient for

estimation concentration range)


In LC-MS one peak, the unknown 1


C42H78N2O7

C34H63NO6

C32H59NO6

C27H53NO3

C29H57NO3

Case 1: Bringing all together

• Note: It is very common to find methyl esters

• Concentration range HTPEP & HTPES low ppb level!


Summary

• LC-HRMS is the technique for unknown identification

• Extractables and Leachables are explainable understanding polymer chemistry and

physicochemical principles

• Unknowns are best identified using material knowledge and chemistry

• Suspected-target screening is the key for fast “unknown” screening

• Quantitative estimations should be performed by authentic reference compounds or

by using orthogonal analytical methods, e.g. UV detection

• “Real” non-target screening is the most time-consuming task and can take months!

“Unknown” screening can make a lot of fun, especially if everything fits togetherThink outside the “even/odd number-nitrogen rule-isotopic patter-fragmentation-mclafferty rearrangement” box.

Enjoy your “Eureka” moment

Contact

Roberto Menzel

Head of Laboratory and Manager Extractables & Leachables

[email protected]

Armin Hauk

Principal Scientist Extractables & Leachables

[email protected]

Website: www.sartorius.com

@SartoriusGlobal

@SartoriusStedimBiotech

Tanja Maier

Scientist Extractables & Leachables

[email protected] Extractable Group Sartorius on ResearchGatehttps://tinyurl.com/y2a4n3rw

Special thanks for data and presentation support

http://www.sartorius.com/


Thank you for your attention!

Question & Discussion

identification of unknowns in lc-ms using a holistic approach · • fragmentation pattern checked...

Documents