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Development of a General Solvents Method for DMSO (ACN) 0.041 2 Anisole 0.5 3 ... p-Xylene 0.0304...

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  • Development of a General Solvents Method for DMSO

    Soluble Compounds

    Anne Warner, Linda Osborne, Robert Riley, V. Robert Stultz

    Lilly Research LaboratoriesEli Lilly & Company

  • Outline

    Goals of Solvent WorkMethod DevelopmentMethod ValidationMethod UseSolvent Control StrategyReference Standards

  • Goals of General Solvent work

    Develop chromatographic conditionsResolve all solvents of interest to process developmentMinimize run timeSimilar to USP/PhEur testsObtain accurate results

    Quantitative Limit test

    Assure sensitivityNo additional method development when applying method to new matrixUse for all steps to monitor/control solvents

  • Solvent listUSP

    or *toxicology Target, %

    Solvent Class

    Acetone (Actn) *0.45 3

    Acetonitrile (ACN) 0.041 2

    Anisole 0.5 3

    Benzene 0.0002 1

    1-Butanol (1-BuOH) *0.05 3

    2-Butanol (2-BuOH) 0.5 3

    Cyclohexane (cyclo) 0.388 2

    1,2-Dichloroethane 0.0005 1

    Diethyl ether *0.1 3

    Dimethylformamide (DMF) *0.04 2

    DMSO 0.5 3

    Ethanol (EtOH) 0.5 3

    Ethyl Acetate (EtOAc) *0.45 3

    Ethyl benzene (etbenz) 0.0369 2

    Heptane (Hept) *0.2 3

    Hexane 0.029 2

    Isobutyl alcohol (2Me-1-PrOH) 0.5 3

    USP or

    *toxicology Target, %

    Solvent Class

    Isopropyl acetate (IPAC) 0.5 3

    Methanol (MeOH) *0.25 2

    t-Methyl butyl ether (MTBE) 0.5 3

    Methyl ethyl ketone (MEK) *0.3 3

    Methyl isobutylketone (MIBK) 0.5 3

    Methylene Chloride (MeCl2) 0.06 2

    N-methylmorpholine 0.1 n/c

    1-Pentanol (amyl alcohol) 0.5 3

    n-Propanol (n-PrOH) 0.5 3

    2-Propanol (IPA) 0.5 3

    Pyridine 0.02 2

    Sulfolane 0.016 2

    Tetrahydrofuran (THF) 0.072 2

    Toluene 0.089 2

    Triethylamine 0.1 n/c

    m-Xylene 0.1302 2

    o-Xylene 0.0195 2

    p-Xylene 0.0304 2

  • Method DevelopmentTemperature program

    ProEzGC Computer based simulation software for gas chromatographyBased on 2 sets of lab runs

    Use same conditions for Direct Injection techniquesHeadspace conditions

    Evaluate vial conditionsTemperatureTimeShake

    Sample solvent/concentrationTarget 100 mg/mL to assure sensitivityMore compounds soluble in DMSO

  • Principle of Head Space GC

    HS VialEquilibrate at a given temperatureVolatile solvents partition between the gas headspace and the sample Partition coefficient, K, is related to the degree of solubility of the analyte in the matrix or solution Cm and the tendency of the analyte to go to the gaseous phase, Cg.

    K = Cm/Cg

    K

    Cg

    Cm

  • Vial ParametersEquilibration Temperature

    Equilibration Time at 85C

    0.00

    100.00

    200.00

    300.00

    400.00

    500.00

    600.00

    55 60 65 70 75 80 85 90 95Temperature, C

    Pea

    k A

    rea Ethanol Peak Area

    Acetone Peak Area

    0

    100

    200

    300

    400

    500

    5 15 25 35 45 55

    Time (min.)

    Pea

    k A

    rea

    Ethanol Peak Area

    Acetone Peak Area

    0

    50000

    100000

    150000

    200000

    75 80 85

    Temperature, C

    Peak

    Are

    a MethanolMethylene Chloride1-butanolDimethylformamide

    0

    50000

    100000

    150000

    200000

    25 30 35

    Time (min)

    Peak

    Are

    a MethanolMethylene Chloride1-butanolDimethylformamide

  • Standard Evaluation

    Area of std alone : Area of std in mixture

    Area of std alone : Area of std in mixture

    ratio, % ratio, %

    Acetone (Actn) 99.6 Methanol (MeOH) 102.4

    Acetonitrile (ACN) 93.3 t-Methyl butyl ether (MTBE) 90.7

    Anisole 100.7 Methyl ethyl ketone (MEK) 104.7

    1-Butanol (1-BuOH) 102 Methylene Chloride (MeCl2) 84.2

    2-Butanol (2-BuOH) 109.2 N-methylmorpholine 109.6

    Cyclohexane (cyclo) 94.5 1-Pentanol (amyl alcohol) 103.9

    Diethyl ether 90.4 n-Propanol (n-PrOH) 105.2

    Ethanol (EtOH) 109.3 2-Propanol (IPA) 88.1

    Ethyl Acetate (EtOAc) 102.1 Tetrahydrofuran (THF) 101.9

    Ethyl benzene (etbenz) 104.6 Toluene 108.3

    Heptane (Hept) 84.7 m-Xylene 93.1

    Hexane 72.9 o-Xylene 109.8

    Isobutyl alcohol (2Me-1-PrOH) 104.3 p-Xylene 93.1

    Isopropyl acetate (IPAC) 103.7

    Evaluation of standard solvent mixture as compared to individual solvent standards for response. (Solvent partitioning is not significantly affected by presence of other solvents in the standard mix)

  • The Method GC ConditionsColumn 6% polycyanopropylphenylsiloxane-

    94% polydimethylsiloxane , for example an Agilent DB-624 or

    equivalent

    Column Dimensions 30 m x 0.32 mm i.d., 1.8 m film thickness

    Carrier Gas Helium

    Flow Rate Approximately 2.1 mL/min

    Injector Liner Direct Inlet Liner, 2mm ID, quartz, deactivated

    Detection Flame Ionization (FID)

    Injector 140C

    Detector Temperature 250C

    Head Pressure Approximately 10 psi

    Split Ratio Approximately 10:1

    Initial Temperature 45C

    Initial Time 5 min.

    Rate 10C/min.

    Final Temperature 175C

    Final Temperature Time

    1 min.

    GC Run Time 19 minutes

  • The Method: Chromatographym

    V

    -50.00

    0.00

    50.00

    100.00

    150.00

    200.00

    250.00

    300.00

    350.00

    400.00

    450.00

    500.00

    550.00

    600.00

    650.00

    700.00

    750.00

    Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

    MeO

    H EtO

    Hdi

    ethy

    l eth

    er

    acet

    one

    IPA

    ACN

    MeC

    l2

    MTB

    E

    hexa

    ne

    n-PrO

    H

    MEK

    EtO

    Ac

    2-BuO

    HTH

    F

    cycl

    ohex

    ane

    isob

    utyl

    alc

    ohol

    isop

    ropy

    l ace

    tate

    hept

    ane

    mV

    - 5 0 .0 0

    0 .0 0

    5 0 .0 0

    1 0 0 .0 0

    1 5 0 .0 0

    2 0 0 .0 0

    2 5 0 .0 0

    3 0 0 .0 0

    3 5 0 .0 0

    4 0 0 .0 0

    4 5 0 .0 0

    5 0 0 .0 0

    5 5 0 .0 0

    6 0 0 .0 0

    6 5 0 .0 0

    7 0 0 .0 0

    7 5 0 .0 0

    Min u te s8 .5 0 9 .0 0 9 .5 0 1 0 .0 0 1 0 .5 0 1 1 .0 0 1 1 .5 0 1 2 .0 0 1 2 .5 0 1 3 .0 0 1 3 .5 0 1 4 .0 0 1 4 .5 0 1 5 .0 0 1 5 .5 0 1 6 .0 0 1 6 .5 0 1 7 .0 0 1 7 .5 0 1 8 .0 0 1 8 .5 0 1 9 .0 0

    1-B

    uOH

    MIB

    Kpy

    ridin

    eto

    luen

    e

    n-m

    ethy

    lmor

    phol

    ine

    amyl

    alc

    ohol

    ethy

    lben

    zene

    m-x

    ylen

    e

    o-xy

    lene A

    niso

    le

    , p-x

    ylen

    e

    DM

    SO

    mV

    - 5 0 .0 0

    0 .0 0

    5 0 .0 0

    1 0 0 .0 0

    1 5 0 .0 0

    2 0 0 .0 0

    2 5 0 .0 0

    3 0 0 .0 0

    3 5 0 .0 0

    4 0 0 .0 0

    4 5 0 .0 0

    5 0 0 .0 0

    5 5 0 .0 0

    6 0 0 .0 0

    6 5 0 .0 0

    7 0 0 .0 0

    7 5 0 .0 0

    Min u te s8 .5 0 9 .0 0 9 .5 0 1 0 .0 0 1 0 .5 0 1 1 .0 0 1 1 .5 0 1 2 .0 0 1 2 .5 0 1 3 .0 0 1 3 .5 0 1 4 .0 0 1 4 .5 0 1 5 .0 0 1 5 .5 0 1 6 .0 0 1 6 .5 0 1 7 .0 0 1 7 .5 0 1 8 .0 0 1 8 .5 0 1 9 .0 0

    1-B

    uOH

    MIB

    Kpy

    ridin

    eto

    luen

    e

    n-m

    ethy

    lmor

    phol

    ine

    amyl

    alc

    ohol

    ethy

    lben

    zene

    m-x

    ylen

    e

    o-xy

    lene A

    niso

    le

    , p-x

    ylen

    e

    DM

    SO

    45C (5min), 10C/min to 175C, 175C (1min)

  • The Method: Chromatography-10% target concentrationm

    V

    0.00

    2.00

    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

    Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

    MeO

    H

    diet

    hyl e

    the r

    IPA

    MeC

    l2

    hexa

    ne

    ME K

    2-Bu

    OH

    cycl

    ohex

    ane

    isob

    utyl

    alc

    ohol

    hept

    ane

    EtO

    H

    acet

    one

    ACN

    MTB

    E

    n-Pr

    OH

    EtO

    Ac

    THF

    isop

    ropy

    l ace

    tat e

    mV

    0.00

    2.00

    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

    Minutes8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00

    MIB

    K

    tolu

    ene

    amyl

    alc

    ohol

    m-x

    ylen

    e

    o-xy

    lene

    1-Bu

    OH

    pyrid

    ine n-

    met

    hylm

    orph

    olin

    e

    ethy

    lben

    zene

    p-xy

    lene

    Anis

    ole

    45C (5min), 10C/min to 175C, 175C (1min)

  • The Method

    Head Space Conditions

    Oven Temperature 85C

    Loop Temperature 95C

    Transfer Line Temp. 130C

    GC cycle time Approximately 25 minutes (adjust as necessary for system cool down and

    equilibration)

    Vial Equilibration Time

    30 minutes

    Pressurize Time 0.5 minutes

    Loop Fill Time 0.2 minutes

    Loop Equilibration Time

    0.1 minutes

    Inject Time 1.0 minutes

    Injection Loop 1 mL

    Vial Pressurization ~ 2.5 psi

    Shake Velocity Low

  • The Method-Standard Addition

    Sample informationPrepare 4 samples to be 100mg/mL when dilutedDilute one with DMSODilute one with standards at the QLDilute one with standards at targetDilute one with standards at 2x target

  • The Method

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