Best Practices for OINDP Pharmaceutical Development Programs Leachables and Extractables VII. Special Case Compound Classes PQRI Leachables & Extractables

Best Practices for OINDP Pharmaceutical Development Programs Leachables and Extractables

VII. Special Case Compound Classes

PQRI Leachables & Extractables Working Group

PQRI Training CourseSeptember 20-21, 2006

Washington, DC

““Special Cases”Special Cases”► PAHs - Polyaromatic HydrocarbonsPAHs - Polyaromatic Hydrocarbons

Also referred to as PNAs (Polynuclear Also referred to as PNAs (Polynuclear Aromatics)Aromatics)

► N-NitrosaminesN-Nitrosamines

► 2-Mercaptobenzothiazole2-Mercaptobenzothiazole

N N OCH3

CH3

S

NSH

September 2006 PQRI Training Course 3

PAHs/PNAs as Leachables in PAHs/PNAs as Leachables in OINDPOINDP

► Historically, the primary source of PNAs is Historically, the primary source of PNAs is carbon carbon black black which is used as a filler in certain types of which is used as a filler in certain types of rubber (mostly sulfur cured).rubber (mostly sulfur cured).

► There is some potential for other PNA sources (e.g. There is some potential for other PNA sources (e.g. naphthalene contamination).naphthalene contamination).

► Some PNAs are known or suspect cancer causing Some PNAs are known or suspect cancer causing agents (e.g. benzo(a)pyrene). FDA interest in MDIs agents (e.g. benzo(a)pyrene). FDA interest in MDIs traces back to the late 1980s.traces back to the late 1980s.

► Levels of PNAs in MDIs which employ “black rubber” Levels of PNAs in MDIs which employ “black rubber” seals are typically on the order of ng to low seals are typically on the order of ng to low µg/canister.µg/canister.

► The FDA historically requires that The FDA historically requires that all elastomersall elastomers in in MDIs be evaluated and controlled for PNAs.MDIs be evaluated and controlled for PNAs.

► Analytical methods typically involve GC/MS.Analytical methods typically involve GC/MS.


PNAs Typically Analyzed and PNAs Typically Analyzed and ControlledControlled

(EPA Method 610 list)(EPA Method 610 list)► NaphthaleneNaphthalene► AcenaphthyleneAcenaphthylene► AcenaphtheneAcenaphthene► FluoreneFluorene► PhenanthrenePhenanthrene► AnthraceneAnthracene► FluorantheneFluoranthene► PyrenePyrene

► Benzo(a)anthraceneBenzo(a)anthracene► ChryseneChrysene► Benzo(b)fluorantheneBenzo(b)fluoranthene► Benzo(k)fluorantheneBenzo(k)fluoranthene► Benzo(e)pyreneBenzo(e)pyrene► Benzo(a)pyreneBenzo(a)pyrene► Indeno(123-cd)pyreneIndeno(123-cd)pyrene► Dibenzo(ah)anthraceneDibenzo(ah)anthracene► Benzo(ghi)peryleneBenzo(ghi)perylene

Structures of Some Typical PNAsStructures of Some Typical PNAs

naphthalenephenanthrene

pyrene

benzo(a)pyrenebenzo(ghi)perylene


Trace Organic AnalysisTrace Organic Analysis


PNA Analysis in Rubber – Possible PNA Analysis in Rubber – Possible MethodMethod

► Slice (or grind) a measured weight of critical Slice (or grind) a measured weight of critical rubber components.rubber components.

► Add prepared rubber to a boiling flask with a Add prepared rubber to a boiling flask with a measured volume of organic solvent (e.g. measured volume of organic solvent (e.g. toluene).toluene).

► Extract via reflux for a pre-optimized time Extract via reflux for a pre-optimized time period (likely 24 hours or greater).period (likely 24 hours or greater).

► Remove solvent and reduce in volume.Remove solvent and reduce in volume.► Analyze by GC/MS (for example).Analyze by GC/MS (for example).

Note that internal standards can be added at Note that internal standards can be added at various points in the overall process.various points in the overall process.


PNA Analysis in a Suspension Metered PNA Analysis in a Suspension Metered Dose Inhaler Drug Product - Possible Dose Inhaler Drug Product - Possible

MethodMethod► Cool sample MDI canisters (one or several for a Cool sample MDI canisters (one or several for a

composite sample) over dry ice.composite sample) over dry ice.► Open canister(s) and filter contents to remove Open canister(s) and filter contents to remove

suspended drug particles.suspended drug particles. Note that filter assembly and catch flask must be cold.Note that filter assembly and catch flask must be cold.

► Wash filter contents with organic solvent.Wash filter contents with organic solvent.► Evaporate sample to dryness.Evaporate sample to dryness.► Dissolve residue in a measured quantity of a Dissolve residue in a measured quantity of a

suitable organic solvent (e.g. toluene).suitable organic solvent (e.g. toluene).► Analyze by GC/MS (for example).Analyze by GC/MS (for example).

Note that internal standards can be added at various Note that internal standards can be added at various points in the overall process.points in the overall process.



A GC/MS SystemA GC/MS System


ReferenceReference

► The information summarized in the following The information summarized in the following slides related to PNA leachables studies is slides related to PNA leachables studies is detailed in the following reference:detailed in the following reference:

Norwood, D.L., Prime, D., Downey, B.P., Creasey, J., Norwood, D.L., Prime, D., Downey, B.P., Creasey, J., Sethi, S.K., Haywood, P., Analysis of polycyclic Sethi, S.K., Haywood, P., Analysis of polycyclic aromatic hydrocarbons in metereds dose inhaler aromatic hydrocarbons in metereds dose inhaler drug formulations by isotope dilution gas drug formulations by isotope dilution gas chromatography/mass spectrometry, chromatography/mass spectrometry, Journal of Journal of Pharmaceutical and Biomedical AnalysisPharmaceutical and Biomedical Analysis, 13(3), , 13(3), 293-304, 1995.293-304, 1995.


GC/MS Analysis of Target PNAsGC/MS Analysis of Target PNAs


EI Spectra of EI Spectra of Pyrene and DPyrene and D1010--

PyrenePyrene

• Note stability of the molecular ions

• Note the characteristic presence of doubly-charged molecular ions


Benzo(e)pyrene and Benzo(a) pyreneBenzo(e)pyrene and Benzo(a) pyrene


Representation LinearityRepresentation Linearity11 and Linearity of Recovery and Linearity of Recovery22 Results for a Drug Product Assay (0.05-2.5µg/inhaler)Results for a Drug Product Assay (0.05-2.5µg/inhaler)

Target PNATarget PNA SlopeSlope InterceptIntercept CorrelationCorrelationNaphthaleneNaphthalene 0.9730.97311 0.0450.045 0.99980.9998

0.9930.99322 0.0330.033 0.99980.9998

AcenaphtheneAcenaphthene 0.6580.658 0.0210.021 0.99980.9998

0.6630.663 0.0140.014 0.99920.9992

PhenanthrenePhenanthrene 1.0591.059 0.0670.067 0.99970.9997

1.0611.061 0.0800.080 0.99900.9990

FluorantheneFluoranthene 1.0701.070 0.0590.059 0.99970.9997

1.0851.085 0.0690.069 0.99960.9996

PyrenePyrene 1.0341.034 0.0590.059 0.99980.9998

1.0401.040 0.0720.072 0.99970.9997

Benzo(e)pyreneBenzo(e)pyrene 1.4851.485 0.0560.056 0.99980.9998

1.4871.487 0.0870.087 0.99940.9994

Benzo(a)pyreneBenzo(a)pyrene 0.8270.827 0.0320.032 0.99960.9996

0.8530.853 0.0540.054 0.99970.9997

Benzo(ghi)peryleneBenzo(ghi)perylene 1.3201.320 -0.016-0.016 0.99960.9996

1.3501.350 -0.028-0.028 0.99980.9998


Limit of Detection/Quantitation Results Limit of Detection/Quantitation Results for Selected Target PNAsfor Selected Target PNAs

Target PNATarget PNA Limit of DetectionLimit of Detection

(ng/inhaler)(ng/inhaler)Limit of QuantitationLimit of Quantitation

(ng/inhaler)(ng/inhaler)

NaphthaleneNaphthalene 0.70.7 44

AcenaphthyleneAcenaphthylene 0.70.7 44

FluoreneFluorene 0.90.9 55

PhenanthrenePhenanthrene 0.90.9 55

FlourantheneFlouranthene 0.70.7 44

PyrenePyrene 0.70.7 44

Benzo(ghi)peryleneBenzo(ghi)perylene 66 3030


PNA Profile of an MDI Drug ProductPNA Profile of an MDI Drug Product


PNAs as Leachables in Metered Dose InhalersPNAs as Leachables in Metered Dose Inhalers

Target PNATarget PNA Product AProduct A(µg/inhaler)(µg/inhaler)

Product BProduct B(µg/inhaler)(µg/inhaler)

Product CProduct C(µg/inhaler)(µg/inhaler)

NaphthaleneNaphthalene 0.290.29 0.150.15 0.570.57

AcenaphthyleneAcenaphthylene 0.430.43 0.220.22 0.450.45

AcenaphtheneAcenaphthene NDND NDND NDND

FluoreneFluorene <0.05<0.05 NDND <0.05<0.05

PhenanthrenePhenanthrene 1.961.96 0.880.88 2.142.14

AnthraceneAnthracene 0.100.10 NDND 0.120.12

FluorantheneFluoranthene 1.201.20 0.530.53 1.371.37

PyrenePyrene 1.261.26 0.610.61 2.132.13

Benzo(a)anthraceneBenzo(a)anthracene NDND NDND NDND

ChryseneChrysene NDND NDND NDND

Benzo(b)fluorantheneBenzo(b)fluoranthene NDND NDND NDND

Benzo(k)fluorantheneBenzo(k)fluoranthene NDND NDND NDND

Benzo(e)pyreneBenzo(e)pyrene 0.080.08 <0.025<0.025 0.080.08

Benzo(a)pyreneBenzo(a)pyrene <0.05<0.05 NDND <0.05<0.05

Dibenzo(ah)anthraceDibenzo(ah)anthracenene NDND NDND NDND

Indeno(123-cd)pyreneIndeno(123-cd)pyrene NDND NDND NDND

Benzo(ghi)peryleneBenzo(ghi)perylene 0.080.08 0.030.03 0.060.06

TotalTotal 5.505.50 2.452.45 7.027.02


N-Nitrosamines as Leachables in N-Nitrosamines as Leachables in OINDPOINDP

► Historically, the formation of “nitrosamines” in Historically, the formation of “nitrosamines” in rubber involves sulfur curing agents (e.g. thiurams).rubber involves sulfur curing agents (e.g. thiurams).

► The issue of N-nitrosamines in rubber goes back to The issue of N-nitrosamines in rubber goes back to late 1970s/early 1980s with concern over their late 1970s/early 1980s with concern over their presence in baby bottle rubber nipples. FDA presence in baby bottle rubber nipples. FDA became involved in the issue. Official analytical became involved in the issue. Official analytical methods for rubber developed and validated.methods for rubber developed and validated.

► FDA interest in MDIs (and other OINDP) traces to FDA interest in MDIs (and other OINDP) traces to the early 1990s.the early 1990s.

► Levels of nitrosamines in MDIs which employ “black Levels of nitrosamines in MDIs which employ “black rubber” seals are typically on the order of rubber” seals are typically on the order of ng/canister.ng/canister.

► The FDA historically requires that The FDA historically requires that all elastomersall elastomers in in MDIs be evaluated and controlled for nitrosamines.MDIs be evaluated and controlled for nitrosamines.

► Analytical methods typically involve GC with Analytical methods typically involve GC with “Thermal Energy Analysis” detection (GC/TEA).“Thermal Energy Analysis” detection (GC/TEA).

Target N-nitrosaminesTarget N-nitrosamines

►N-nitrosodimethylamineN-nitrosodimethylamine►N-nitrosodiethylamineN-nitrosodiethylamine►N-nitrosodi-n-butylamineN-nitrosodi-n-butylamine►N-nitrosomorpholineN-nitrosomorpholine►N-nitrosopiperidineN-nitrosopiperidine►N-nitrosopyrrolidineN-nitrosopyrrolidine

N N OCH3

CH3

O

N

N

O

N

N

O

N

N

O


N-nitrosamine FormationN-nitrosamine Formation

NOX N N O

CH3

CH3

HXN

CH3

CH3

H + +

N

S

CH3

CH3

S

N

S

CH3CH3

S N

CH3

CH3

HS

N

S

CH3CH3

CS2+heat

.

H .

X = NO+, N2O3, N2O4, NOZ (Z = Cl, Br, I, thiocyanate)


N-nitrosamine Analysis in RubberN-nitrosamine Analysis in Rubber(AOAC Method 987.05)(AOAC Method 987.05)

► Place 5g cut rubber sample in 250mL flask with 100mL Place 5g cut rubber sample in 250mL flask with 100mL methylene chloride and 100mg propyl gallate, and hold for methylene chloride and 100mg propyl gallate, and hold for 17-18h.17-18h.

► Transfer solvent and rubber sample to a Transfer solvent and rubber sample to a Soxhlet extractorSoxhlet extractor..► Spike in internal standard.Spike in internal standard.► Extract for 1 hour.Extract for 1 hour.► Add 100mL 5N NaOH and 2g Ba(OH)Add 100mL 5N NaOH and 2g Ba(OH)2 2 to flask and carefully to flask and carefully

distill distill methylene chloride (discard). Continue distilling 70mL methylene chloride (discard). Continue distilling 70mL of aqueous distillate into a of aqueous distillate into a sepratory funnelsepratory funnel..

► Add 300mg anhydrous NaAdd 300mg anhydrous Na22COCO33 to funnel, followed by 50mL to funnel, followed by 50mL methylene chloride. methylene chloride. ExtractExtract (repeat twice more). Combine (repeat twice more). Combine extracts in sepratory funnel.extracts in sepratory funnel.

► Pass through anhydrous NaPass through anhydrous Na22SOSO44 (to dry), into a (to dry), into a Kuderna_Danish apparatus (with appropriate washes).Kuderna_Danish apparatus (with appropriate washes).

► ConcentrateConcentrate to approximately 4mL. to approximately 4mL.► Remove from KD and further concentrate to 1.0mL with a Remove from KD and further concentrate to 1.0mL with a

nitrogen stream.nitrogen stream.► Analyze by GC/TEA.Analyze by GC/TEA.


N-nitrosamine Analysis in RubberN-nitrosamine Analysis in Rubber(AOAC Method 987.05)(AOAC Method 987.05)

Soxhlet extractionSteam distillation

Extract concentration

Image provided by Rubber Consultants


Principles of Thermal Energy Analysis Principles of Thermal Energy Analysis DetectionDetection

► N-nitrosamines elute from a GC column into a N-nitrosamines elute from a GC column into a pyrolyzer, where they undergo pyrolysis and release pyrolyzer, where they undergo pyrolysis and release nitrosyl radicals (NOnitrosyl radicals (NO..). The pyrolysis temperature is ). The pyrolysis temperature is set low enough so that nitro-compounds will not set low enough so that nitro-compounds will not pyrolyze.pyrolyze.

► Nitrosyl radicals are then oxidized with ozone in a Nitrosyl radicals are then oxidized with ozone in a reaction chamber to give electronically excited NOreaction chamber to give electronically excited NO22

**..► The NOThe NO22

* * decays back to ground state releasing a decays back to ground state releasing a photon at a characteristic wavelength.photon at a characteristic wavelength.

► This process is known as “chemiluminescence”.This process is known as “chemiluminescence”.► Sensitivity is further increased through use of a Sensitivity is further increased through use of a

filter-photometer for detection.filter-photometer for detection.


A GC/TEA System – Schematic DiagramA GC/TEA System – Schematic Diagram

GC

pyrolysis

450oCcold trap

-130oC

ozone

vacuum

detector

electronics

0 2 4 6 8 10Time (min)

6000

8000

100001200014000160001800020000

µV

N-d

imeth

yla

min

e

N-m

eth

yle

tthyla

min

e

N-d

ieth

yla

min

e

Inte

rnal sta

ndard

(N

DIP

A)

N-d

ipro

pyla

min

e

N-d

ibuty

lam

ine

N-p

iperi

din

e

N-p

yrr

olidin

e

N-m

orp

holine


A GC/TEA SystemA GC/TEA SystemImage provided by Rubber Consultants


A GC/TEA SystemA GC/TEA System

Image provided by Cardinal Health


GC/TEA N-nitrosamines - SeparationGC/TEA N-nitrosamines - Separation

0 2 4 6 8 10Time (min)

6000

8000

1000

012

000

1400

016

000

1800

020

000

µV

N-d

imet

hyla

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e

N-m

ethy

lett

hyla

min

e

N-d

ieth

ylam

ine

Inte

rnal

sta

ndar

d (N

DIP

A)

N-d

ipro

pyla

min

e

N-d

ibut

ylam

ine

N-p

iper

idin

e

N-p

yrro

lidin

e

N-m

orph

olin

e


GC/TEA N-nitrosamines - SensitivityGC/TEA N-nitrosamines - Sensitivity

0 2 4 6 8 10Time (min)

6000

8000

1000

0µ

V

N-d

ieth

ylam

ine

N-m

ethy

leth

ylam

ine

N-d

ieth

ylam

ine

Inte

rnal

sta

ndar

d (N

DIP

A)

N-d

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N-d

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N-p

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N-p

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N-m

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10 ng/mL


Some Typical Limit of Some Typical Limit of Detection/Quantitation Results for Detection/Quantitation Results for

Target N-nitrosaminesTarget N-nitrosamines

►AOAC Method 987.05 LOQs target AOAC Method 987.05 LOQs target acceptance criteria of NMT 10ppb acceptance criteria of NMT 10ppb (ng/g) for an individual N-nitrosamine.(ng/g) for an individual N-nitrosamine.

►Based on the LOQs for rubber, MDI Based on the LOQs for rubber, MDI methods should target LOQs around 1 methods should target LOQs around 1 ng/canister. ng/canister.


N-nitrosamines in OINDP – Points to N-nitrosamines in OINDP – Points to ConsiderConsider

►N-nitrosamines are usually associated N-nitrosamines are usually associated with sulfur-cured black rubber.with sulfur-cured black rubber.

►Even with the sensitivity and selectivity Even with the sensitivity and selectivity of the GC/TEA, other peaks are often of the GC/TEA, other peaks are often noted in OINDP leachables profiles.noted in OINDP leachables profiles.

►N-nitrosamines are very light sensitive, N-nitrosamines are very light sensitive, which suggests a possible procedure which suggests a possible procedure for identifying “non-nitrosamine” for identifying “non-nitrosamine” GC/TEA peaks.GC/TEA peaks.


Analysis of Mercaptobenzothiazole (MBT) Compounds from Sulfur Cured Rubber by a

Liquid Chromatography – Tandem Mass Spectrometry (LC-MS-MS) Method

Tianjing Deng*, Shuang Li, Xiaoya Ding and Song Klapoetke

PPD

8551 Research WayMiddleton, WI 53562

* Corresponding author


• Mercaptobenzothiazole (MBT) and other benzothiazoles are

common vulcanization accelerators for rubber materials that are used in pharmaceutical container/systems, such as the gaskets in the pressurized Metered-Dose Inhaler (pMDI). MBT is of particular concern since it is considered a potential carcinogen and has been shown to migrate into drug formulations.

• Due to the toxicological concern and leachability of MBT and other

benzothiazoles, analytical methods have been developed to study these types of compounds in the fields of food additives and contaminants (1), contact dermatitis caused by the rubbers (2), as well as pharmaceutical packaging systems (3). MBT can be analyzed by gas chromatography (4) but many other benzothiazoles are thermally-labile and readily decomposed in the GC inlet. HPLC methods are commonly used to study


In this study, a method using liquid chromatography with tandem mass spectrometer (LC-MS-MS) was developed to analyze MBT in the sulfur cured rubber. The method is capable of detecting ng level of MBT in the rubber extracts. This study demonstrates the feasibility of using detector with high selectivity, such as LC-MS-MS method, for extractable/leachable with special toxicological concern that requires greater sensitivity and specificity.

S

NSH

S

NS

S

NS

MBT MBTS


RReferences: 1Barnes, K.A., Castle L., Damant, A. P., Read, W. A., and Speck, D. R., Food Additives and Contaminants, Vol. 20, No. 2, 196-205 (2003).2Hansson, C., Bergendorff, O., Ezzelarab, M., and Sterner, O., Contact Dermatitis, 36, 195-200, (1997).3Gaind, V. S., and Jedrzejczak, K., Journal of Analytical Toxicology, Vol. 17, 34-37, (1993).4Niessen, W. M. A., McCarney, C. C., Moult, P. E.G., Tjaden, U. R., and Van der Greef, J., Journal of Chromatography, 647, 107-119, (1993).5Mathieu, C., Herbreteau, B., Lafosse, M., Morin, Ph., Renaud, M., Cardinet, C., and Dreux, M., J. High Resol. Chromatogr., 23, (9), 565-566, (2000).6


Method Conditions:

HPLC Parameters

Mobile Phase: Water:Methanol:Formic acid 20:80:0.05 (v/v/v)Flow Rate: 0.2 mL/minColumn: Waters Symmetry C18, 3.5 m, 2 x 100 mmColumn Temperature: 40°CAutosampler Temperature: AmbientInjector Volume: 5 L


““Bench-top” LC/MS SystemsBench-top” LC/MS Systems

Linear ion trap

Triple Quadrupole

Time-of-flight


Triple Quadrupole Mass Triple Quadrupole Mass SpectrometerSpectrometer


+Product (168.0): 64 MCA scans from Sample 1 (direct) of MBT2.wiff Max. 1.4e7 cps.

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220m/z, amu

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

Inte

ns

ity, c

ps

168.1

135.1

124.1

136.2

92.2109.2

110.2

65.1141.180.2 104.1 169.177.1 95.2

S

N

SH

Mercaptobenzothiazole (MBT)

MS-MS Spectrum of MBT


+Product (333.0): 84 MCA scans from Sample 2 (MBTs) of MBT2.wiff Max. 5.6e5 cps.

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

Inte

ns

ity, c

ps

167.0

333.2

257.2198.0

S

N

S

S

N

S

Dibenzothiazyl Disulfide (MBTS)

MS-MS Spectrum of MBTS


Mass Spectrometer:

PE Sciex API 2000/API365 Triple Quadruple Mass Spectrometer

Ionization Mode: Positive ElectroSpray Detection Mode: MRM MBT @ m/z 168/135 MBTS @m/z 333/167


1 2 3 4 5 6 7 8 9 10 11 12 Time (min)

0

5

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15

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25

30

35

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45

50

55

60

65

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6.09

MRM Chromatogram of Extraction Blank: MBT (blue trace) and MBTS (red trace)

Selectivity/Specificity


MRM Chromatograms of MBT (blue) and MBTS (red) in MRM Chromatograms of MBT (blue) and MBTS (red) in the 500 ng/mL standard solution.the 500 ng/mL standard solution.

XIC of +MRM (2 pairs): 168.0/135.0 amu from Sample 5 (STD-NF) of DataSET2.wiff Max. 5.7e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4 5.7e4 1.11

MBT

MBTS


MRM Chromatograms of MBT (blue) and MBTS MRM Chromatograms of MBT (blue) and MBTS (red) in the 30 min TBME Extract.(red) in the 30 min TBME Extract.

XIC of +MRM (2 pairs): 168.0/135.0 amu from Sample 22 (30min-1) of DataSET1.wiff Max. 8.9e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

8.9e4

Inte

ns

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ps

1.11


Extraction Method

Hansson et al. studied the extraction of MBT/MBTS using different solvents. They found out that Methyl tert-Butyl Ether (MTBE) is a good solvent for MBT/MBTS due to its:

• Powerful extraction medium

• Low toxicity

• Inertness to MBT/MBTS

• High volatility

In this study, the rubber was cut into 3 x 3 mm squares. One gram of the rubber was extracted with 10 mL MTBE for 30 minutes by sonication. After extraction, the extract was diluted to different volume using Methanol: Water 50: 50 diluent to give varying MBT concentrations and filtered using glass fiber syringe filters for LC-MS study.


020406080

100120140160180200

5 10 20 30 40 50

Extraction Time

Ext

ract

aed

MB

T (

pp

m)

The Extraction Study of MBT by MTBE from Sulfur-Cured Rubber


0

200000

400000

600000

800000

1000000

1200000

0 200 400 600 800 1000 1200

Concentration (ng/mL)

Res

po

nse

MBT

MBTS

Linearity Plot of MBT (50 – 1000 ng/mL)


Repeatability

Calculated MBT Concentration (PPM) in Three Replicates of Extract.

Calculated Concentration (PPM) Mean %RSD (n=3)

Replicate 1 Replicate 2 Replicate 3

153.4 157.1 149.6 153.4 2.4


LOQ/LOD

1 2 3 4 5 6 7 8 9 10 11 12

Time (min)

0

20

40

60

80

100

120

140

160

180

200

220

240

259

4.83 MBT

MRM Chromatogram of MBT Standard (50 ng/mL)

The DL of MBT was calculated using S/N ratio = 3. DL = 6 ng/mL in the solution or 12 pg on column.


Accuracy - Filter Study

A Filter study was conducted to verify that the syringe filter used in the sample preparation did not reduce the recovery of MBT and MBTS. Three 500 ng/mL standards were analyzed before and after the filtration and the area responses of MBT and MBTS were compared. The percent differences between the filter and non-filtered samples are less than 2.5% indicating that filtration does not affect the method accuracy.

Mean area responses Compounds MBT MBTS

Before Filtration 4082 2130 After Filtration 3990 2151 % Difference 2.3 1.0


Accuracy- MBT Recovery

Approximately 360 ng/mL of MBT was spiked into the extract. The sample was prepared using the sample preparation procedure and analyzed. Three replicates of

spiking samples were prepared and analyzed. The mean recovery of MBT was 87.3%

Recovery Results of MBT

Extract(ng/mL)

Replicate 1(ng/mL)

Replicate 2(ng/mL)

Replicate 3(ng/mL)

Calculated219.5 567.8 552.6 553.2

%RSD 3.5 (n=7) 2.3 2.4 3.0

%Recovery NA 89.9 86.0 86.1


Summary PointsSummary Points

►The developed LC/MS/MS method The developed LC/MS/MS method looks good for MBT, and potentially looks good for MBT, and potentially MBTS.MBTS.

►MBTS was demonstrated to hydrolyze MBTS was demonstrated to hydrolyze under the extraction conditions under the extraction conditions selected, forming more MBT.selected, forming more MBT.

►This method requires full optimization This method requires full optimization and validation.and validation.


Analytical Method ValidationAnalytical Method Validation

► System suitabilitySystem suitability Chromatographic parameters (e.g. resolution, tailing)Chromatographic parameters (e.g. resolution, tailing) Injection precisionInjection precision

► PrecisionPrecision RepeatabilityRepeatability Intermediate PrecisionIntermediate Precision

► SelectivitySelectivity► Accuracy (three spiking levels)Accuracy (three spiking levels)► Linearity/RangeLinearity/Range► LOD/LOQLOD/LOQ► Robustness (e.g. column, mobile phase, Robustness (e.g. column, mobile phase,

temperatures, MS parameters)temperatures, MS parameters)


Concluding PointsConcluding Points

►““Special Case” compounds require Special Case” compounds require dedicated and highly specific dedicated and highly specific analytical methods.analytical methods.

►Dedicated and highly specific Dedicated and highly specific analytical methods have been analytical methods have been developed for all “special case” developed for all “special case” compounds and compound classes.compounds and compound classes.

Documents

Best Practices for OINDP Pharmaceutical Development Programs Leachables and Extractables VII. Special Case Compound Classes PQRI Leachables & Extractables