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 sensitivity☺No additional method development when applying method to new matrixUse for all steps to monitor/control solvents

Solvent listUSP<467>

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<467> 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 85˚C

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 µm 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 140ºC

Detector Temperature 250°C

Head Pressure Approximately 10 psi

Split Ratio Approximately 10:1

Initial Temperature 45°C

Initial Time 5 min.

Rate 10°C/min.

Final Temperature 175°C

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

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

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

The Method

Head Space Conditions

Oven Temperature 85°C

Loop Temperature 95°C

Transfer Line Temp. 130°C

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

System Suitability3 injections of standard at the target

RSDarea <20%Rs closest peaks >1.0

Use the sample spiked with the QL standard to verify S/N >10

Rs = 1.0*

*Figure from: Dolan, J.W.; Snyder, L.R. “Troubleshooting LC Systems”, 1989, Humana Press, NJ

The Method Standard Addition Calculations

Calibration curve (area vs. conc.) for each solvent

Using: unspiked, QL, target, 2 x target

slopeintercept-y mg/mL conc,solvent =

(%) 100 x mg/mL 100

mg/mLconc,solvent % conc,solvent =

Standard addition

0

500

1000

1500

2000

2500

-0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3

analyte concentration, mg/mL

anal

yte

area

in s

ampl

e m

atrix

}

Method Validation

Evaluation of standardsSpecificity

Absence of background interferenceIdentifiable peaks, Rs≥1.0

LinearityRange evaluated: 10-200% of target limitNumber levels: 6Number of replicates/level: 3Results: range for r = 0.998 to 1.000

Method Validation, con’t

RepeatabilityRange evaluated: 10-200% of target limitNumber levels: 6Number of replicates/level: 3Results, range for %RSD: 0.07% - 3.67%

QLQL is defined as 10% of target limit (lowest point evaluated)At the QL, S/N ≥ 10

Example Compound ATwo solvents potentially present

EtOAc, THF Performed test 3 times

Replicate1 Replicate2 Replicate3

Slope 1485.617 1449.996 1510.728

y-Intercept 723935.3 724115.1 706393

Result, % 0.49 0.50 0.47

Replicate1 Replicate2 Replicate3

Slope 2743.09 2705.384 2763.106

y-Intercept 28368.37 27718.82 26692.16

Result, ppm 103 102 97

0

500000

1000000

1500000

2000000

EtO

AC

are

a

-250 0 250 500 750 1000EtOac conc

0

100000

200000

300000

400000

500000

THF

area

0 50 100 150THF conc

Example Compound B

Analyte Slope Interceptppm

Result

MeOH 776869 2514 <250

EtOAc 3387238 -9552 <450

THF 6068225 -3358 <72

pyridine 33835 50 <20

DMF 110231 56 <40

Five solvents potentially presentNo analyte detected in unspiked sampleS/N>10 for sample spiked at 10% target

EtOAc

-10000000

1000000200000030000004000000

0 0.2 0.4 0.6 0.8 1

Level of spike in mg/mL

Res

pons

e

MeOH

0100000200000300000400000500000

0 0.1 0.2 0.3 0.4 0.5 0.6

Level o f sp ike in mg / mL

Res

pons

eTHF

-500000

0

500000

1000000

0 0.05 0.1 0.15

Level of spike in mg/mLR

espo

nse

Pyridine

0

500

1000

1500

0 0.01 0.02 0.03 0.04 0.05

Level of spike in mg/mL

Res

pons

e

DMF

0

10000

20000

30000

0 0.05 0.1 0.15 0.2

Level of spike in mg/mL

Res

pons

e

Comparison to compendia

DMI- 1,3-dimethyl-2-imidazolidinone used for DMF determinations*the temperature depends on the sample solvent

^no conditions described to determine when to use what parameters

PhEur USP Lilly general

sample solventwater/DMF/DMI/other demonstrated suitable water/DMF DMSO

concentration, mg/mL 10 10 100standard solvent DMSO then water DMSO then water/water DMSOvial equilibration Temp, °C 80/105/80/as demonstrated suitable* 80/105/80/as demonstrated suitable^ 85vial equilibration time, min 60/45/45/as demonstrated suitable 60/45/45/as demonstrated suitable 30transfer line Temp, °C 85/110/105/as demonstrated suitable 85/110/105/as demonstrated suitable 130Carrier gas Nitrogen or Helium Nitrogen or Helium HeliumPressurization time, sec 30 30 30Injection volume, mL 1 1 1

column phase6% polycyanopropylphenylsiloxane :

94% polydimethylsiloxane 6% polycyanopropylphenylsiloxane :

94% polydimethylsiloxane 6% polycyanopropylphenylsiloxane :

94% polydimethylsiloxane

column dimensions30m x 0.32mm (or 0.53mm) x

1.8um (or 3um)30m x 0.32mm (or 0.53mm) x

1.8um (or 3um) 30m x 0.32mm x 1.8umsplit ration 5:1 5:1 10:1linear velocity cm/s ~35 ~35 ~35detector FID (/MS/ECD) FID FIDdetector temp, °C 250 250 250injection temperature, °C 140 140 140

Temperature program40°C (20min), 10°C/min to 240°C,

240°C (20min) 40°C (20min), 10°C/min to 240°C,

240°C (20min) 45°C (5min), 10°C/min to 175°C,

175°C (1min) Run time 60 min 60 min 19 min

standardsall class 1 and 2 solvents and

relevant class 3 solventsclass 1 and 2 stds- USP RS mix

(class 3 by LOD/valid procedure) all relevant solvents

System suitability

S/N(1,1,1 trichloroethane) ≥ 5 Rs (ACN,MeCl2) ≥ 1.0,

chromatography resembles Figure

S/N(1,1,1 trichloroethane) ≥ 5 S/N(class 1 solvents) ≥ 3 Rs (ACN,MeCl2) ≥ 1.0,

RSD of smallest peak ≤ 20% (n=3) Rs(closest peaks) ≥ 1.0 S/N(smallest

pk in detectability sample) ≥ 10no pk for class 1 or 2 solvent in

sample: passsample peak < class 1 or 2 std pk:

passpk for class 1 or 2 present in sample,

confirm with PEG columnpk for class 1 or 2 present in sample,

confirm with PEG columnstds spiked individually into samples stds combined

result format pass / fail limit test qualitative/semi-quantitative quantitative testuses matrix assumes no matrix effect uses matrix

AdvantagesStandard addition eliminates need to validate every matrix individually

Validation exists every time method is run

Demonstrating method suitability every timeNo need for additional comparison of results to compendial methodsNo change in GC setup required

Efficiency gain

Overall control strategy for processStarting materialsIntermediatesS l t d i

Issues

Sample SolubilitySome initial solubility information is needed

If not DMSO soluble then determine what is a good solventUse similar chromatographic conditions if possible

DMSO qualityPreferred supplier that is reliable

Reactivity of some samples with DMSONot enough sensitivity for every analyteat 1/10 target

benzene and 1,2 DCEUse a limit test method with same chromatographic conditions

Also issue with high boiling point solvents

Additional topics

Routine GC testing may not be requiredNo testingSkip testing

Determine long-term strategy for solvent reference standards…some options

Prepare by analystPrepare by vendor (Restek, Supelco, etc)Maintained by corporate ref std groupMaintained from USP

Reference Standard Recommendations

Maintained standard locally in laboratory Utilize external vendor for tailored solvent mixtures

Fully characterized standards with certificate of analysis to assure correct standard potencyNo shipping issuesStandard material will be available when needed