A Fast and Sensitive Method for Residual Hydrazine Analysis in
Pharmaceutical Samples
Vera Leshchinskaya, Priscilla Richberg, Yury Zelechonok*, and Anne KellyBristol Myers Squibb Company, P.O.BOX 4000, Princeton, NJ, 08543,* SIELC Technologies, 65 E. Palatine Rd., Suite 221, Prospect Heights, IL 60070
Hydrazine Properties
Hydrazine isa highly reactive molecule widely used in chemical syntheses of intermediates and active pharmaceutical ingredients a known carcinogen - can have an impact on product risk assessment if present in the final drug substance and drug product as an impurity. a small, polar, basic molecule with no chromophore
NH2 NH2
Objective
Develop a method for detection of low (ppm) level of hydrazine in pharmaceutical samples and wash solutions. Method criteria:
simple sample preparation stepefficiencysensitivityease of transfer
Control of Genotoxic Impurities in Pharmaceuticals
According to current regulatory guidance, it is assumed that genotoxic compounds have the potential to damage DNA at very low level of exposure. Thus, actual and potential impurities most likely to arise during synthesis, purification, and storage should be identified. Both the EMEA guidelines and a PhRMA white paper propose a limit of 1.5 µg/day for genotoxic impurities in pharmaceuticals.
Analytical Challenges in Setting Limits for Genotoxic Impurities
Low level (ppm) needs to be detectedMany genotoxic impurities are highly reactive and therefore difficult to analyzeSample matrix can be chemically similar and present at extremely high level Techniques being employed aren’t standard and may present significant technology transfer issues
Analytical Background
Existing methods for hydrazineare mostly applicable to environmental samples (water,air,soil)employ sophisticated equipment and/or derivatizationtend to be cumbersome and time consumingare subject to interference, especially by hydrazine derivatives (colorimetric and titrimetric methods)
GC Analysis of Residual Hydrazine
NH2 NH2 + O
CH3
CH3
2 N
NCH3
3
CH3
CH3
CH
Advantages of using Acetone:Serves as both diluent and derivatizing agentSymmetrical molecule (produces only one isomer)Fast reaction rateLow toxicityGood GC separation from the product of the reaction.
GC Analysis of Acetone Azine
2.53
1A
ceto
ne a
zine
- 2.
822
3.27
4
3.70
8
pA
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
220.00
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Oven Program:Initial temperature: 95°C
15.251.0095.040.03
11.004.00225.030.02
3.500.00120.010.01
TotalTime
HoldTime
FinalRate
Ace
tone
Ace
tone
impurity
Column:20-m length x 0.18-mm i.d., 1µm film thickness, 6% cyanopropylphenyl-94% dimethylpolysiloxane (DB-624); Detector: FID, 280°C; Injector: 1 μL into the split port maintained at 200 °C;Carrier Gas: Helium; Column Flow: 1.3 ml/min; Split ratio/Split Flow: 110/143 ml/min.
Linearity Data for Acetone Azine
y = 105.48x + 0.0297R2 = 0.9999
01020
3040506070
8090
100
0 0.2 0.4 0.6 0.8 1Concentration (mg/mL)
Peak
Are
a
S/N at 0.84 ppm is ~10:10.84 ppm of Acetone azinecorresponds to 0.24 ppm of free hydrazine in solution or 2.4 ppm/100 mg of sample
88.3914842.00.842
54.1033505.20.5052
8.809784.20.0842
5.265850.50.05052
0.83848.400.00842
0.50775.050.005052
0.11680.840.000842
ppmmg/mL Peak Area
Concentration
Conversion of Hydrazine Mono-Hydrate to Acetone Azine: GC/MS Data
NH2 NH2 + O
CH3
CH3
2N
NH2CH3
CH3
N
NCH3
CH3
CH3
CH3
MW=72 MW=112
100806040m/z
11297
958070
67
56
523928
11010090807060504030m/z
7257
544139
3130
acet
one
- 1.0
07
mon
o-su
bstit
uted
- 2.
010
Ace
tone
azi
ne -
2.88
5
pA
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50
Conversion Rate of Hydrazine Mono-Hydrate to Acetone Azine
Rate of Conversion of Hydrazine to Acetone azine
0
50
100
150
200
250
300
350
400
0 200 400 600 800
Time, min
Are
a co
unts
Acetone Azine
Propane-2-ylidenhydrazine
Time, min %Conversion5 10.3
103 22.8153 29.1170 31.7187 34.1204 36.8259 37.5276 39.7573 69.1590 77.0
Reaction Conversion of Hydrazine Mono-Hydrate to Acetone Azine
Catalyzed by Acid
0/1000/10015
5.5/94.50/1000
CH3COOHHCOOH
% Conversion, Acetone Hydrazone/ Acetone Azine
Time, min
Conclusion : The derivatization proceeds in less than 3 min in the presence of formic acid and within ~15 min with Acetic Acid.
Reaction Conversion of Hydrazine Dihydrochloride to Acetone Azine
Problem: Low solubility of the salt in acetoneConversion rate is ~50% within 24 hours
Solution:Standard prep
- dissolved~ 10 mg/2 ml of H2O- diluted with acetone to 200 mlThe derivatization proceeds in 2.5 hours(97.5%). Presence of formic acid did not show improvement in the reaction rate
Sample Analysis for Residual Hydrazine.
Problem:~100 mg needed to be dissolved in 1 ml of acetone for ppmlevel hydrazine analysis.
Solution:Sample prep
- added 1 ml of 1/99 H2O/Acetone to 100 mg of the sample.
- Heated at~ 40oC. Mixed well. The solution became clear for a short period of time (1-2 minutes)and then the precipitate formed again under cooling to room temperature.
Question:Did the reaction occur or did the recrystallizationtake place?
N
NHNH 2
* HCl
Compound A
1H NMR Spectra Comparison (1)
0.011.021.030.990.991.002.74
DMSO-d6
2.49
3.15
6.896.
926.937.02
7.16
7.17
7.28
7.31
7.41
7.427.
967.
967.
979.41
10.0
0
13 12 11 10 9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)
1.95
2.00
2.03
2.04
2.07
2.12
2.16
2.496.
926.94
6.95
7.23
7.24
7.38
7.46
7.47
7.49
7.67
7.697.
827.
83
11.2
5
Compound A
Precipitate from Acetone, yield is ~100%
N
NHN H 2
* HCl
N
NHN
C H 3CH 3
DMSO-d6, 400 MHz
1H NMR Spectra Comparison (2)
DMSO-d6, 400 MHz
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2Chemical Shift (ppm)
1.66
1.751.78
1.82
1.97
2.10
2.15
2.496.916.93
6.94
7.18
7.22
7.23
7.37
7.45
7.477.67
7.697.82
7.83
11.1
211
.13
11.1
411
.15
11.1
6
Precipitate from Acetone spiked with Acetone Azine (AA)
Acetone layer
AA
NMR Study of reaction conversion
NMR of the precipitate showed mostly hydrazone, the conversion is close to100% by weightNMR of supernatant showed ~30% of unreacted sample, hydrazone and acetone azine formed due to the presence of residual hydrazine in the sampleSpiking experiment showed 100.9% recovery of spiked hydrazine (synergistic effect?)
Residual Hydrazine Analysis in Neutral Molecules with Low Solubility in Acetone
Procedure:- Dissolved sample (~50mg) in NMP or any
other suitable solvent- Added 0.1 ml of acetone/HCOOH; - Analyzed sample by the method described
aboveThe recovery of hydrazine from solutionfortified with hydrazine mono-hydrate was103%
Direct Analysis of Hydrazine in AqeousSolutions
3.09
9
11.0
64
mV
0.00
200.00
400.00
600.00
800.00
1000.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
3.09
9
-3.00
-2.00
-1.00
0.00
1.00
2.00
3.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00Column: Primesep 100 (Sielc Technology)Gradient: 20%ACN/80%H2O/0.2%TFA for 6 min, then 80%ACN/20%H2O/0.2%TFA, 1 ml/min, Sample concentration - 31.4 mg/ml, inj. Vol.- 10 µlDetection: ELSD; T= 40OC; Photomultiplier voltage- 500
Mixed-Mode Chromatography:
Direct Analysis of Hydrazine by Mixed-Mode Chromatography
Calibration Curve for Hydrazine Dihydrochloride
y = 159952x1.3903
R2 = 0.9992
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
0 2 4 6
Y
mg/ml
ELSDCalibration Curve for Hydrazine
Monohydrate
y = 892671x - 6081.7R2 = 0.9972
0.0E+00
2.0E+05
4.0E+05
6.0E+05
8.0E+05
1.0E+06
1.2E+06
1.4E+06
1.6E+06
1.8E+06
0 0.5 1 1.5 2
mg/ml
CAD
peak
Are
a
CAD
Detection limit - ~100 ppm Detection limit - ~35 ppm
Conclusion
The reaction of acetone with hydrazine free base and HCl salts has been investigated and conditions were developed for quantitative and fast conversion to acetone azineA fast and sensitive GC method for residual hydrazine analysis in pharmaceutical samples has been developedExcellent linear relationship between acetone azine concentration and FID response was demonstrated with minimal detectable concentration 0.24 ppmThe method is applicable to pharmaceutical compounds at all stages of development with some modificationsHydrazine can be analyzed in aqueous solutions using mixed-mode chromatography and CAD detection with minimal detectable concentration 35 ppm
Acknowledgments
Su Pan (CAD data)Kana YamamotoReginald CannXinhua Qian