A Fast and Sensitive Method for Residual Hydrazine Analysis in Pharmaceutical Samples Vera Leshchinskaya, Priscilla Richberg, Yury Zelechonok*, and Anne Kelly Bristol Myers Squibb Company, P.O.BOX 4000, Princeton, NJ, 08543, * SIELC Technologies, 65 E. Palatine Rd., Suite 221, Prospect Heights, IL 60070 Hydrazine Properties H2N NH2 Hydrazine is a 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 Objective Develop a method for detection of low (ppm) level of hydrazine in pharmaceutical samples and wash solutions. Method criteria: simple sample preparation step efficiency sensitivity ease 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 detected Many genotoxic impurities are highly reactive and therefore difficult to analyze Sample 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 hydrazine are mostly applicable to environmental samples (water,air,soil) employ sophisticated equipment and/or derivatization tend to be cumbersome and time consuming are subject to interference, especially by hydrazine derivatives (colorimetric and titrimetric methods) GC Analysis of Residual Hydrazine CH3 CH3 H3C N H N NH 2 O 2 2 + N CH3 CH3 H3C Advantages of using Acetone: Serves as both diluent and derivatizing agent Symmetrical molecule (produces only one isomer) Fast reaction rate Low toxicity Good GC separation from the product of the reaction. GC Analysis of Acetone Azine 220.00 2 82 . Oven Program: 200.00 2 Initial temperature: 95°C ne - 180.00 i az e impurity on 160.00 one Rate Final Hold Total et et c Time Time A Ac 140.00 1 10.0 120.0 0.00 3.50 8 70 120.00 e 3. 2 30.0 225.0 4.00 11.00 A p on 100.00 et 3 40.0 95.0 1.00 15.25 Ac 80.00 60.00 40.00 4 27 531 20.00 3. 2. 1.00 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 Minutes 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 100 Concentration y = 105.48x + 0.0297 90 2 mg/mL ppm Peak Area R = 0.9999 80 0.000842 0.84 0.1168 0.005052 5.05 0.5077 70 ea 0.00842 8.40 0.8384 r 60 0.05052 50.5 5.2658 0.0842 84.2 8.8097 eak A 50 P 0.5052 505.2 54.1033 40 0.842 842.0 88.3914 30 20 S/N at 0.84 ppm is ~10:1 10 0.84 ppm of Acetone azine corresponds to 0.24 ppm 0 of free hydrazine in solution 00.20.40.60.81or 2.4 ppm/100 mg of sample Concentration (mg/mL) Conversion of Hydrazine Mono-Hydrate to Acetone Azine: GC/MS Data CH3 CH 3 H C N H3C NH2 3 H2N NH2 2 O + N N CH3 CH 3 MW=72 H C MW=112 H3C 3 7 57 72 160.00 00 30 31 1. 39 140.00 41 54 one - 30 40 50 60 70 80 90 100 110 120.00 et m/z c a 100.00 010 pA 80.00 885 56 ed - 2. 97 60.00 ut 39 112 t 70 i 28 ne - 2. t i 52 67 80 95 40.00 ubs 40 60 80 100 s m/z 20.00 one az et ono- c m 0.00 A 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes Conversion Rate of Hydrazine Mono- Hydrate to Acetone Azine Rate of Conversion of Hydrazine to Acetone azine Time, min %Conversion 5 10.3 400 Acetone Azine 103 22.8 350 153 29.1 300 170 31.7 187 34.1 s 250 t n u 204 36.8 200 259 37.5 ea co r A 150 276 39.7 100 573 69.1 50 590 77.0 Propane-2-ylidenhydrazine 0 0 200 400 600 800 Time, min Reaction Conversion of Hydrazine Mono-Hydrate to Acetone Azine Catalyzed by Acid Time, % Conversion, min Acetone Hydrazone/ Acetone Azine HCOOH CH3COOH 0 0/100 5.5/94.5 15 0/100 0/100 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 acetone Conversion rate is ~50% within 24 hours Solution: Standard prep - dissolved~ 10 mg/2 ml of H2O - diluted with acetone to 200 ml The 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. NH2 Problem: H N ~100 mg needed to be dissolved in 1 ml of acetone for ppm * HCl N level hydrazine analysis. Solution: Sample prep Compound A - 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 recrystallization take place? 1H NMR Spectra Comparison (1) H 3 C CH3 NH2 H N N H N DMSO-d6, 400 MHz * HCl N N DMSO-d62.16 2.12 2.49 7.02 6.93 9.41 6.92 7.17 7.16 7.97 7.96 7.96 Compound A 7.28 7.42 7.41 6.89 7.31 10.00 3.15 7.47 2.74 1.00 0.990.991.031.02 0.01 7.46 7.23 2.07 7.67 7.82 7.24 7.83 6.94 7.69 6.95 6.92 7.49 7.38 Precipitate from Acetone, 2.49 yield is ~100% 11.25 2.04 2.03 2.00 1.95 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 16 15 1 14 H NMRSpectra Comparison(2) 13 with Acetone Azine Precipitate from Acetonespiked 12 Acetone layer 11.16 11.15 11 11.14 11.13 11.12 10 9 DMS 7.83 Chemical Shift(ppm) 7.82 8 7.69 7.67 ( 7.47 O 7.45 AA) 7.37 - 7 7.18 7.23 d 7.22 6, 400MHz 6.94 6.93 6 6.91 5 4 3 2.49 2.15 2.10 2 1.97 1.82 1.78 1.75 1.66 1 AA 0 -1 -2 NMR Study of reaction conversion NMR of the precipitate showed mostly hydrazone, the conversion is close to100% by weight NMR of supernatant showed ~30% of unreacted sample, hydrazone and acetone azine formed due to the presence of residual hydrazine in the sample Spiking 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 above The recovery of hydrazine from solution fortified with hydrazine mono-hydrate was 103% Direct Analysis of Hydrazine in Aqeous Solutions 1000.00 Mixed-Mode 800.00 064 Chromatography: 3.00 11. 600.00 2.00 mV 400.001.00 099 3. 0.00 200.00 -1.00 099 3. 0.00 -2.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 -3.00 Minutes Column: Primesep 100 (Sielc Technology) Gradient: 20%ACN/80%H2O/0.2%TFA2.00 4.00 for6.00 6 8.min,00 10. 0then0 12. 00 14.00 16.00 18.00 20.00 80%ACN/20%H2O/0.2%TFA, 1 ml/min, Sample concentration - 31.4 mg/ml, inj. Vol.- 10 µl Detection: ELSD; T= 40OC; Photomultiplier voltage- 500 Direct Analysis of Hydrazine by Mixed-Mode Chromatography ELSD CAD Calibration Curve for Hydrazine Calibration Curve for Hydrazine Dihydrochloride Monohydrate 1.8E+06 1600000 y = 892671x - 6081.7 y = 159952x1.3903 1.6E+06 R2 = 0.9972 1400000 R2 = 0.9992 1.4E+06 1200000 a 1.2E+06 e 1000000 Ar 1.0E+06 k a e Y 800000 8.0E+05 6.0E+05 600000 CAD p 4.0E+05 400000 2.0E+05 200000 0.0E+00 0 00.511.52 mg/ml 0246 mg/ml 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 azine A fast and sensitive GC method for residual hydrazine analysis in pharmaceutical samples has been developed Excellent linear relationship between acetone azine concentration and FID response was demonstrated with minimal detectable concentration 0.24 ppm The method is applicable to pharmaceutical compounds at all stages of development with some modifications Hydrazine 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 Yamamoto Reginald Cann Xinhua Qian.