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
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