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