PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE COMPOUND − ACCEPTANCE TESTING AND FORMS

PET Radiopharmaceutical Sciences Section,Date of review: 12/01/05

Molecular Imaging Branch,

National Institute of Mental Health,

National Institutes of Health,

Bldg. 10, Rm. B3 C338,

Bethesda, MD20892

1.Overview

The radiopharmaceutical production of [C-11]-PBR 28 for the National Institute of Mental Health requires the use of desmethyl PBR 28 ( 3-[N-Acetyl-N-(2-hydroxybenzyl)amino]-4-phenoxypyridine) as a precursor for C-11 methylation. The standard operating procedure (SOP) for acceptance testing of desmethyl PBR 28 is described below. Acceptance testing is required to validate chemical purity and chemical identity of desmethyl PBR 28. Upon acceptance of desmethyl PBR 28, the desmethyl PBR 28 may be released for the production of [C-11]-PBR 28. Upon release, the desmethyl-PBR 28 is to be tested annually thereafter for chemical purity (via HPLC, section 3.1) and chemical identity (via LC-MS, section 3.2).

This SOP also contains testing methods and acceptance criteria for a standard sample of PBR 28 (section 5). This non-radioactive material is utilized in the quality control testing of [C-11]-PBR 28 for the determination of specific radioactivity and chemical identity.

2.Synthetic Schemes for Preparation of Reference Standard, PBR 28 and precursor compound, Desmethyl PBR 28

PBR 28 is synthesized according to Nakazoto A. et al. Japaneses Patent, 2000/001476, as outlined in Scheme A. Desmethyl PBR 28 is produced in six steps from commercially available salicylaldehyde, 4-chloro-5-nitro-pyridine and phenol, according to Scheme B.

Scheme A. Synthesis of PBR28 from 4 and commercial material (10).

Scheme B. Synthesis of desmethyl PBR28 from commercial materials (1,2 and 5)

3. Acceptance criteria for precursor and reference compound

(A) Precursor

Source: the starting material, desmethyl PBR 28 is obtained from this laboratory.

Acceptance criteria: the acceptance criteria for precursor are as follows:

  • 1H-NMR spectrum of desmethyl PBR 28 from this laboratory to verify chemical structure.
  • LC-MS of desmethyl PBR 28 performed inthe PET Radiopharmaceutical Sciences Section on the Finnigan LCQDeca LC/MS(ThermoQuest, Germany) to confirm chemical structure and purity
  • HPLCin the PET Radiopharmaceutical Sciences Section showing the chemical purity of precursor to be ≥ 98%.

(B) Reference compound

Source: the reference compound (PBR 28) is obtained from this laboratory.

Acceptance criteria: the acceptance criteria for reference compound are as follows:

  • 1H-NMR spectrum of PBR 28 from this laboratory to verify chemical structure.
  • LC-MS of PBR 28 performed in the PET Radiopharmaceutical Sciences Section on the Finnigan LCQDeca LC/MS (ThermoQuest, Germany) to confirm chemical structure and purity
  • HPLC in the PET Radiopharmaceutical Sciences (MIB, NIMH, NIH) showing the chemical purity of PBR 28 to be ≥ 98%.

4. Precursor and reference compounds acceptance forms

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH

DesmethylPBR 28 Acceptance Form

Receipt of compound:

Compound name: Desmethyl PBR 28 / Lot #
Supplier: PET Radiopharmaceutical Sciences Section (PRSS), Molecular Imaging Branch (MIB)
National Institute of Mental Health
National Institutes of Health (NIH)
Building 10, Room B3C338
10 Center Drive, MSC 1003
Bethesda, MD20892 / Received by:
Date received: / Quantity:
Molecular formula:C20H18N2O3 / Molecular weight:334.37

1H-NMR spectrum

Record weight of desmethyl PBR 28 ______dissolved in ca. 0.8 mL of 0.03% TMS (v/v) in CDCl3

Proton Assignment / Expected Resonance (ppm) / Observed Resonance (ppm) / Acceptance?
CO-CH3 / 2.03 (s, 3H) / Y or N
methylene / 4.82 (dd, 2H) / Y or N
Aromatic protons / 8.44 (d, 1H, ), 8.39 (s,1H), 7.40-7.42 (m, 2 H), 7.30-7.35 ( m, 1H), 7.28 (Solvent, CDCl3), 7.25-7.27 (m, 1H), 6.95-6.92 (d, 1H), 6.65-6.77 (m, 5H) / Y or N

DesmethylPBR 28 Acceptance Form

HPLC

Record weight ______mg in 10 ml of 20/80 Ethanol/saline.

Column: Onyx (monolithic C18; 4.6 mm x 100 mm; Phenomenex; part # CH0 7643)
Eluent: Isocratic: 0.01 M ammonium formate in water-acetonitrile (70: 30 v/v)
Flow rate: 3 mL/min
UV Detector: 220 nm

Record Serial # of this column: ______

Criterion / Acceptance? (circle one) / Date
Retention time 4.5251.0 min / Y or N
Peak area ≥ 98% / Y or N

LC/MS

Criterion (expected m/z, M + 1) / Observed ion, M + 1 / Acceptance (circle one)?
335.13 / Y or N

Attached documents, acceptance criteria and test results:

1.1H-NMR spectrum from this laboratory? YES______NO______.

2. LC-MS data from this laboratory?YES______NO______.

3. HPLC chromatogram from this laboratory? YES______NO______.

(An example of a set of satisfactory attached documents is included in Document 8).

DesmethylPBR 28 Acceptance Form

Chemical structure:
/ Acceptance criteria:
  1. Identity: confirmed by 1H-NMR and LC/MS
  2. Purity: ≥ 98% by HPLC
  3. Identity: confirmed by LC/MS

Test Results
Test / Results / Procedure / Date / Initials
1. NMR ( This laboratory) / SOP # QA308(2.1)
2. LC-MS (This laboratory) / SOP # QA308(2.3)
3. HPLC (This laboratory) / SOP # QA308(2.2)
Checked by: / Accepted by:
Storage condition: / Date
PBR 28 Reference Acceptance Form

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH

Receipt of compound:

Compound name: PBR 28 / Lot #
Supplier Name: PET Radiopharmaceutical Sciences Section (PRSS), Molecular Imaging Branch (MIB)
National Institute of Mental Health
National Institutes of Health (NIH)
Building 10, Room B3C338
10 Center Drive, MSC 1003
Bethesda, MD20892 / Received by:
Date received: / Quantity:
Molecular formula: C21H20N2O3 / Molecular weight: 348.4

1H-NMR spectrum

Record the weight ______of PBR 28 dissolved in ca. 0.8 mL of 0.03% TMS (v/v) in CDCl3

Proton Assignment / Expected Resonance (ppm) / Observed Resonance (ppm) / Acceptance?
-CO- CH3 / 2.01 (S, 3H) / Y or N
-OCH3 / 3.60 (S, 3H) / Y or N
-CH2 / 5.18- 5.14 (dd, 2H) / Y or N
Aromatic protons / 8.30 (d, 1H), 8.23 (S, 1H), 7.28 (Solvent, CDCl3), 7.46~7.22 (m, 5H), 6.59~6.93 ( m, 5H), / Y or N

PBR 28 Reference Acceptance Form

HPLC

Record weight of reference compound _____ mg in 10 ml of 20/80 USP grade ethanol/ saline.

Column: Onyx (monolithic C18; 4.6 mm x 100 mm; Phenomenex; part # CH0 7643)

Eluent: Isocratic: 0.01 M ammonium formate in water-acetonitrile (70: 30 v/v)

Flow rate: 3 mL/min

UV Detector: 220 nm

Record Serial # of this column: ______

Criterion / Acceptance? (circle one) / Date
Retention time 6.683min / Y or N
Peak area≥ 98% / Y or N

LC/MS

Criterion (expected m/z, M + 1) / Observed ion, M + 1 / Acceptance (circle one)?
349.15 / Y or N

Attached documents, acceptance criteria and test results:

1.1H-NMR spectrum from this laboratory attached? YES______NO______.

2. LC-MS data from this laboratory?YES______NO______.

3. HPLC chromatogram from this laboratory? YES______NO______.

(An example of a set of satisfactory attached documents is included in Document 8).

PBR 28 Reference Acceptance Form

Chemical structure:
/ Acceptance criteria:
  1. Identity: confirmed by 1H-NMR
  2. Purity: ≥ 98 % by HPLC
  3. Identity: confirmed by LC/MS

Test Results
Test / Results / Procedure / Date / Initials
1. NMR ( This laboratory) / SOP # QA308(3.1)
2. LC-MS (This laboratory) / SOP # QA308(3.3)
3. HPLC (This laboratory) / SOP # QA308(3.2)
Checked by: / Accepted by:
Storage condition: / Date

Chemist______Initials______Date______

Document 8. [11C]PBR 28for Injection: Precursor and Reference Compound − Acceptance Testing and Form Page 1 of 16