What mass should an AOD-9604 certificate show?

PubChem lists AOD-9604 with an average molecular weight of 1815.1 g/mol and a monoisotopic mass of 1813.86 Da, both for the disulfide-bonded form. Spectra usually show multiply charged ions, so the report should give a deconvoluted mass. A result close to these figures supports identity; one lower by about a tyrosine residue suggests the shorter AOD9401 fragment.

Is AOD-9604 the same as AOD9401?

No. AOD9401 is human growth hormone residues 177 to 191. AOD-9604 adds a tyrosine at the N-terminus, making 16 residues instead of 15. The extra residue changes the formula and mass, so a certificate written for one does not confirm the other. Mass spectrometry tells them apart easily if the expected mass is set correctly.

How does a test show the disulfide bond is intact?

The bonded and open forms differ by two hydrogen atoms, which accurate mass can detect. A stronger check is peptide mapping under reducing and non-reducing conditions: if Cys7 and Cys14 are joined, the fragments containing them stay linked until the bond is reduced. ICH guidance names both approaches for products with expected disulfide bridges.

Can AOD-9604 form dimers in the vial?

It can. Each molecule has two cysteines, and if they pair with cysteines on a neighbouring molecule instead of with each other, the result is a covalent dimer at roughly twice the expected mass. Dimers and oligomers are a recognised impurity class in synthetic peptides, so the mass range of an identity test should reach high enough to see them.

One of the 30 compound testing profiles

AOD-9604

AOD-9604 is a 16-amino-acid peptide (C78H123N23O23S2, average mass 1815.1 g/mol) closed by a disulfide bond between Cys7 and Cys14; a test should confirm that mass, show that the ring is intact rather than reduced or cross-linked into dimers, and rule out the shorter fragment sold under a similar code.

AOD-9604 is a synthetic 16-residue peptide made of the C-terminal fragment of human growth hormone from residues 177 to 191 with an extra tyrosine added at the N-terminus (Cox et al., 2015).

Test your AOD-9604

Identity card

Formula: C78H123N23O23S2
Average mass: 1,815.1 g/mol
Monoisotopic mass: 1,813.86 Da
Length: 16 residues
Modifications: Disulfide bond Cys7-Cys14; Free N-terminus; Free C-terminal acid
Also sold as: AOD9604, Tyr-hGH(177-191), YLRIVQCRSVEGSCGF
Sequence: YLRIVQCRSVEGSCGF
Note: PubChem's formula and masses describe the disulfide-bonded form, named in its record as the (7->14)-disulfide. Residues 2 to 16 correspond to human growth hormone residues 177 to 191.

Source: Aod-9604 compound record (CID 71300630).

Compare the mass printed on a certificate with the values above. The mass checker explains charge states and which mass a lab usually reports.

What usually goes wrong with AOD-9604. Documented failure modes, with sources.

AOD9604 versus AOD9401: one tyrosine apart

Two closely related codes appear in the literature. AOD9401 was described as the lipolytic domain of human growth hormone, residues 177 to 191, made by solid-phase synthesis (Ng et al., 2000). AOD9604 is the same fragment with an additional tyrosine at the N-terminus (Cox et al., 2015), which PubChem records as an N-tyrosyl analogue (PubChem). The two differ by one tyrosine residue, a gap any working mass spectrometer resolves, provided the report compares against the right expected mass.

Reduced, open-chain material instead of the ring

AOD-9604's structure depends on a single disulfide between Cys7 and Cys14. If that bond is not formed, or is broken, the peptide is an open chain with two free thiols and two more hydrogen atoms than the PubChem formula. In mass spectrometry a disulfide-linked species is assigned by a mass 2 Da below the sum of its free-thiol parts, and disulfide reduction and scrambling are monitored as critical quality attributes of protein drugs (Lakbub et al., 2018). A low-resolution identity result can blur a 2 Da gap.

Disulfide-linked dimers and oligomers

Free cysteines on two different AOD-9604 molecules can join each other instead of closing their own ring, creating covalent dimers. Dimeric to oligomeric impurities are one of the recognised classes of impurity in synthetic peptide medicines (D'Hondt et al., 2014). In forced-degradation work on oxytocin, another peptide with one internal disulfide, disulfide- and dityrosine-linked dimers formed under stress, along with tri- and tetrasulfide forms of the monomer (Hawe et al., 2009). A dimer has roughly twice the monomer mass and is missed if the scanned mass range is too narrow, so a report should state the range it covered.

Incomplete side-chain deprotection on a 16-residue chain

AOD-9604 carries several residues whose side chains are protected during synthesis, including Tyr1, Arg3 and Arg8, Gln6, Ser9 and Ser13, Glu11 and both cysteines. Inefficient removal of side-chain protection leaves peptide-protection adducts, and unprotected side chains can react with synthesis reagents (D'Hondt et al., 2014). An adduct is heavier than the target by the mass of what stayed attached, so mass data identify it; a purity figure only shows that something else is present.

Which tests matter most

LC-MSConfirms the 16-residue sequence with its tyrosine, distinguishing AOD-9604 from AOD9401 and other growth hormone fragments, and detects dimers.
HRMSAccurate mass separates the disulfide-bonded form from the reduced open chain, which differs by only two hydrogen atoms.
Peptide mappingComparing digests under reducing and non-reducing conditions confirms that Cys7 and Cys14 are joined to each other.
HPLC-UVShows the share of the main peak and separates adducts, truncated chains and dimers for mass identification.

Reading AOD-9604 results

PubChem gives AOD-9604 an average molecular weight of 1815.1 g/mol and a monoisotopic mass of 1813.86 Da (PubChem). The report should state the deconvoluted neutral mass, not just an observed m/z value (reading a mass spectrum), and the mass checker compares it with both PubChem values.

ICH guidance for biotechnological products says that where cysteines are expected, the number and positions of free sulfhydryl groups and disulfide bridges should be determined, and that peptide mapping under reducing and non-reducing conditions or mass spectrometry may be useful (ICH Q6B). AOD-9604 is synthetic rather than biological, but a certificate that confirms the disulfide, not just the overall mass, answers the harder question.

Public Tested.ph results

No public AOD-9604 records yet

Each public AOD-9604 record will appear here with its identity, HPLC purity and measured amount against the label, once the first vial is tested.

FIG 13Amount against the label

Questions about testing AOD-9604

What mass should an AOD-9604 certificate show?: PubChem lists AOD-9604 with an average molecular weight of 1815.1 g/mol and a monoisotopic mass of 1813.86 Da, both for the disulfide-bonded form. Spectra usually show multiply charged ions, so the report should give a deconvoluted mass. A result close to these figures supports identity; one lower by about a tyrosine residue suggests the shorter AOD9401 fragment.
Is AOD-9604 the same as AOD9401?: No. AOD9401 is human growth hormone residues 177 to 191. AOD-9604 adds a tyrosine at the N-terminus, making 16 residues instead of 15. The extra residue changes the formula and mass, so a certificate written for one does not confirm the other. Mass spectrometry tells them apart easily if the expected mass is set correctly.
How does a test show the disulfide bond is intact?: The bonded and open forms differ by two hydrogen atoms, which accurate mass can detect. A stronger check is peptide mapping under reducing and non-reducing conditions: if Cys7 and Cys14 are joined, the fragments containing them stay linked until the bond is reduced. ICH guidance names both approaches for products with expected disulfide bridges.
Can AOD-9604 form dimers in the vial?: It can. Each molecule has two cysteines, and if they pair with cysteines on a neighbouring molecule instead of with each other, the result is a covalent dimer at roughly twice the expected mass. Dimers and oligomers are a recognised impurity class in synthetic peptides, so the mass range of an identity test should reach high enough to see them.

Sources

Aod-9604 compound record (CID 71300630), PubChem, US National Library of Medicine
Cox HD et al. Detection and in vitro metabolism of AOD9604. Drug Test Anal. 2015;7(1):31-8, Drug Testing and Analysis (PubMed)
Ng FM et al. Molecular and cellular actions of a structural domain of human growth hormone (AOD9401) on lipid metabolism in Zucker fatty rats. J Mol Endocrinol. 2000;25(3):287-98, Journal of Molecular Endocrinology (PubMed)
Lakbub JC et al. Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins. Anal Bioanal Chem. 2018;410(10):2467-2484, Analytical and Bioanalytical Chemistry (PMC)
D'Hondt M et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30, Journal of Pharmaceutical and Biomedical Analysis (PubMed)
Hawe A et al. Towards heat-stable oxytocin formulations: analysis of degradation kinetics and identification of degradation products. Pharm Res. 2009;26(7):1679-88, Pharmaceutical Research (PMC)
ICH Q6B: Specifications, test procedures and acceptance criteria for biotechnological/biological products, International Council for Harmonisation

Last reviewed 6 Oct 2026.