Identity is confirmed by orthogonal evidence, not by a purity figure. A mass measurement places the molecule at 390.14 monoisotopic; MS/MS fragmentation or amino acid analysis fixes the sequence Ala-Glu-Asp-Gly; a chromatogram run on a phase that actually retains a small polar peptide places it against a reference standard.
- Epithalon is the tetrapeptide Ala-Glu-Asp-Gly, formula C14H22N4O9, average mass 390.35 and monoisotopic mass 390.1387.
- Area-percent HPLC purity is a ratio within one chromatogram and carries no information about chemical identity.
- The sequence contains no aromatic residue, so detection at 280 nm is meaningless and purity must be measured near 214 nm.
- The Asp-Gly bond generates an iso-aspartyl isomer that is mass-identical to the parent and can only be resolved chromatographically.
- No FDA or EMA approved product contains AEDG and no pharmacopoeial reference standard exists, so identity evidence must be built from the lot data itself.
Identity and purity answer different questions
A certificate that says Purity: 99.2% (HPLC) and nothing else has told you about the shape of one chromatogram. Area-percent purity is a ratio calculated inside a single trace: the integrated area of the peak someone nominated as the product, divided by the total integrated area of everything the detector saw. It carries no information about what that peak is. Substitute a different tetrapeptide of similar polarity and the number does not move.
ICH Q6B makes the point in regulatory language. An identity test has to be highly specific and based on unique aspects of the molecular structure. Purity, content and identity are three separate determinations, and in peptide work they are answered by three different kinds of measurement. Confusing them is the single most common failure in the research-chemical paperwork I have read.
Epithalon makes the distinction sharper than most compounds, for two reasons that come straight out of its structure. It is very small and very polar, which means an ordinary reversed-phase method may not retain it at all. And it contains an Asp-Gly bond, which generates an isomeric degradant that no mass measurement will ever separate from the parent. Both of those are covered below.
The molecule, in the terms a certificate should use
Epithalon is the tetrapeptide alanyl-glutamyl-aspartyl-glycine, written AEDG in one-letter code and Ala-Glu-Asp-Gly in three-letter. It is a free acid at both termini: no C-terminal amide, no N-terminal acetyl. That leaves one basic site (the N-terminal primary amine) against three carboxylates (Glu side chain, Asp side chain, C-terminal Gly). Net charge at neutral pH is negative, and that governs how it behaves in both the chromatograph and the source.
| Parameter | Value | Note |
|---|---|---|
| Sequence | Ala-Glu-Asp-Gly (AEDG) | Free acid, unmodified termini |
| Synonyms | Epitalon, epithalone, AEDG peptide | Spelling drifts; sequence does not |
| CAS number | 307297-39-8 | Cross-check against PubChem before accepting a printed number |
| Molecular formula | C14H22N4O9 | Free-acid form, salt-free basis |
| Average molecular weight | 390.35 g/mol | The number to use for weighing and molarity |
| Monoisotopic mass | 390.1387 Da | The number an accurate-mass spectrum reports against |
| [M+H]+ | 391.146 | Positive-mode ESI; often weak for this molecule |
| [M-H]- | 389.131 | Negative mode frequently gives the better response |
| [M+Na]+ | 413.128 | Common, and commonly misreported as an impurity |
| Chromophore | None above about 230 nm | No Trp, Tyr or Phe; 280 nm detection is meaningless here |
That last row is a useful screening test on the paperwork itself. Epithalon contains no aromatic residue, so it has no absorbance at 280 nm worth measuring. A certificate reporting HPLC purity at 280 nm for AEDG was either generated on a different compound or copied from a template.
What mass spectrometry settles, and what it cannot
An accurate-mass measurement is the fastest strong evidence available. If a high-resolution instrument returns 391.1459 for the protonated species within a few parts per million, and the isotope pattern matches C14H22N4O9, you have constrained the elemental composition tightly. Low-resolution single-quadrupole data reporting "391" constrains it much less; several plausible synthesis by-products land within a nominal unit.
What mass cannot do is distinguish isomers. Everything in the following list has the same molecular formula and the same exact mass as Epithalon:
- Any sequence permutation of the same four residues, for example Gly-Asp-Glu-Ala or Ala-Asp-Glu-Gly.
- The iso-aspartyl variant, in which the Asp side-chain carboxyl rather than the alpha-carboxyl forms the bond to glycine.
- Any diastereomer arising from racemization at Ala, Glu or Asp during synthesis.
The iso-aspartyl case is not hypothetical. Asp-Gly is the classic aspartimide-forming motif in solid-phase synthesis and in aged peptides: the Asp side chain cyclizes onto the following backbone nitrogen to give a succinimide, which then reopens to either the normal alpha-linked peptide or the beta-linked isoAsp form. The succinimide intermediate is one water lighter, so it does show as a distinct mass at 373.135 for [M+H]+. The reopened isoAsp product does not. It is mass-identical, and separating it needs chromatography with enough resolution to see two closely eluting peaks, or a peptidase-based assay.
Mass does catch the coarser synthesis failures, which is why it earns its place. Deletion variants read cleanly: des-Gly gives [M+H]+ 334.125, des-Ala gives 320.109. Incomplete side-chain deprotection shows as +56.06 for a retained tert-butyl ester. Acetylation adds 42.011. One trap in this particular sequence: the des-Ala tripeptide at 320.109 has the same nominal and exact mass as the y3 fragment of the intact peptide, so an operator reading a survey scan that includes in-source fragmentation can talk themselves into an impurity that is not there. The distinction lies in whether the signal appears in MS1 without collision energy applied.
Sequence confirmation by fragmentation
Tandem MS turns a mass into a sequence claim. Collision-induced dissociation of the precursor generates b and y ions along the backbone, using the Roepstorff and Fohlman nomenclature that has been standard since 1984. For a four-residue peptide the expected set is short enough to read off a printed spectrum without software.
| Ion | Composition | m/z | Typically observed |
|---|---|---|---|
| [M+H]+ | AEDG | 391.146 | Precursor |
| b2 | Ala-Glu | 201.087 | Yes |
| b3 | Ala-Glu-Asp | 316.114 | Yes, often strong |
| y1 | Gly | 76.040 | Low mass, frequently below the scan floor |
| y2 | Asp-Gly | 191.066 | Yes |
| y3 | Glu-Asp-Gly | 320.109 | Yes |
A spectrum showing b2, b3, y2 and y3 covers every amide bond and rules out sequence permutation. That is a genuine identity test in the ICH Q6B sense. Note that fragmentation still says nothing about stereochemistry, and nothing definitive about alpha versus beta linkage at the Asp-Gly bond, since isoAsp b and y ions differ only in the ratio of certain secondary fragments rather than in their nominal positions.
Where MS/MS is unavailable, amino acid analysis after acid hydrolysis is the older orthogonal route. It returns the molar ratio of Ala, Glu, Asp and Gly, which for this peptide should be near 1:1:1:1, and it also gives peptide content on a mass basis. It cannot order the residues. Combining composition from AAA with a retention-time match to an authenticated standard is the usual substitute, and it is weaker than fragmentation data. Chiral confirmation, if a protocol requires it, means derivatizing the hydrolysate with a chiral reagent such as Marfey's reagent and separating the resulting diastereomers, or chiral GC-MS. Almost no research-grade certificate includes this, and few applications need it, but silence on the point should be read as silence rather than as a pass.
Chromatography for a very polar tetrapeptide
This is where certificates for Epithalon go wrong most often. On a conventional C18 column with 0.1% trifluoroacetic acid in water and acetonitrile, a four-residue peptide carrying three carboxylates has almost nothing to hold onto. It elutes at or very near the void volume, along with the salts, the residual scavengers and any inorganic carryover from lyophilization. A peak sitting at 1.1 minutes in a 25-minute gradient is not a purity determination. It is a statement that everything polar in the vial came off the column at once.
Methods that do retain it exist. Hydrophilic interaction chromatography on an amide or zwitterionic phase is the common choice. Mixed-mode phases with an anion-exchange component work on the carboxylates directly. Polar-endcapped or aqueous-stable C18 phases run at high water content give some retention. Porous graphitic carbon is another option for small polar analytes. Whichever is used, the certificate should name the column, the mobile phases, the gradient, the flow rate, the column temperature and the detection wavelength, and the attached trace should have a legible time axis with the injection marked.
Detection is the second constraint. With no aromatic residue, quantitation has to run on the amide bond absorbance in the 210 to 220 nm region. That band is shared by every peptidic species in the sample, which is convenient for relative comparison and unhelpful for anything without an amide. Mannitol, sucrose, glycine used as a bulking agent, sodium chloride: all effectively invisible at 214 nm, or nearly so. A vial that is 40% bulking agent by mass can return 99% area purity honestly. Common practice for a defensible number is a retention factor of at least 2 for the main peak, adequate resolution from the nearest impurity, and a mass balance that reconciles the chromatographic purity against separately measured water, counterion and residual solvent.
Reading the certificate
Run through the document in this order. It takes about four minutes and catches most of what is catchable from paperwork alone.
| Check | What good looks like | What it catches |
|---|---|---|
| Lot linkage | Lot number on every attached spectrum and chromatogram, matching the vial label | Generic certificates recycled across lots |
| Sequence stated | Ala-Glu-Asp-Gly written out, plus formula C14H22N4O9 | Compounds identified only by trade synonym |
| Mass reported correctly | Observed m/z with the adduct and charge named, and whether average or monoisotopic | "MW 390.35 [M+H]+" and similar category errors |
| MS/MS or AAA present | Fragment table or molar ratio, not just a single precursor mass | Sequence permutation and isomeric material |
| HPLC method disclosed | Column, mobile phase, gradient, wavelength in the 210 to 220 nm range | Purity numbers from methods that never retained the analyte |
| Retention sanity | Main peak well clear of the void, integration table shown | Void-volume elution reported as purity |
| Water and counterion | Karl Fischer or TGA, plus acetate or TFA content | Gross weight treated as peptide weight |
| Dates and signature | Analysis date, analyst or laboratory, method reference | Documents that were never generated by an instrument |
There is no pharmacopoeial reference standard for AEDG that I am aware of, and no USP monograph. That matters for how the evidence has to be assembled: you cannot lean on a retention-time match against an authoritative standard the way you can for a compendial article. Identity has to be built from within the data, which in practice means accurate mass plus fragmentation, with chromatography carrying the purity and homogeneity burden. Our quality standard page sets out which of these tests accompanies a lot as a matter of course and which are available on request.
Three red flags are worth naming. Purity quoted to two decimal places with no chromatogram attached. A mass spectrum reproduced as a single stick with no axis or no isotope envelope. And identical retention times across lots reported to the second, which happens on real instruments about as often as identical fingerprints.
Content, counterion and the arithmetic that follows
Once identity is settled, the number that governs bench work is net peptide content rather than gross fill weight. A lyophilized tetrapeptide arrives as some combination of peptide, counterion, bound water and whatever excipient was in the pre-lyophilization solution. Epithalon has only one basic site, so the counterion load from a TFA or acetate salt is modest by mass compared with a heavily basic peptide, but water uptake is not modest at all. Small, highly carboxylated peptides are hygroscopic.
Worked example. A vial labelled 50 mg gross, with a certificate reporting 88% net peptide content, contains:
50 mg × 0.88 = 44 mg peptide
Brought into 2 mL of diluent, that is:
44 mg ÷ 2 mL = 22 mg/mL peptide
The 25 mg/mL figure obtained from the gross weight is 14% high. In molar terms, using the average mass of 390.35 g/mol, 22 mg/mL is approximately 56 mM. Whether that difference matters depends on the protocol, and the point of writing both numbers into the record is that a later reader can tell which basis was used. The vial concentration calculator does the same arithmetic for other fills, and cost per mg is worth running on a net rather than gross basis when comparing suppliers, since a 15% moisture difference moves the real price by 15%.
Diluent choice is a documentation item too. For a peptide with three carboxyl groups, the pH of the reconstituting solvent affects both solubility and the rate of the Asp-Gly aspartimide route, and unbuffered water will not sit where you assume it sits. The note on bacteriostatic water covers what that diluent is and is not.
Regulatory position
Epithalon (AEDG) is not an active ingredient in any product approved by the FDA or the EMA. It is not the subject of a USP monograph, and no pharmacopoeial reference standard exists for it. Most of the published work on the tetrapeptide originates from Russian-language research groups from the late 1990s onward, and that literature has not been the basis of a marketing authorization in the United States or the European Union.
Practically, that means there is no compendial identity procedure to follow and no official standard to compare against. The evidentiary burden sits entirely with the supplying laboratory and the receiving one. It also means claims about what the compound does are outside anything a certificate of analysis can support, and outside the scope of this page.
Status verified 26 August 2026.
FOR LABORATORY AND IN-VITRO RESEARCH USE ONLY. NOT FOR HUMAN OR ANIMAL CONSUMPTION. NOT FOR PERSONAL, MEDICAL, DIAGNOSTIC, THERAPEUTIC, OR RECREATIONAL USE.
What is Epithalon studied for?
Published research on Epithalon investigates the areas below — which is a different question from what Epithalon will do for anyone, a claim about a living system that nothing on this site is sold for.
What it is. A man-made peptide of four building blocks.
What the research looks at. It appears in a limited set of animal and cell studies. The evidence behind it is far thinner than the volume of commercial writing about it suggests.
How it is thought to work. Not established. The published record is small, and much of the original work comes from a narrow group of researchers, which is a real limit when reading it.
What is not established. No approved product, no official standard, and no settled mechanism. Claims made for this compound in vendor copy routinely go well past what the sources they cite actually say.
The full record, including the certificate for the lot in stock, is on the Epithalon product page.
Common questions
Does a high HPLC purity figure confirm that a vial contains Epithalon?
Why is 280 nm detection a warning sign on an Epithalon certificate?
What is the iso-aspartyl variant and why does mass spectrometry miss it?
Which single test gives the strongest identity evidence?
Why would a normal C18 method fail for this compound?
Is net peptide content the same as purity?
Sources
- ICH Q6B, Specifications for Biotechnological and Biological Products. Establishes that an identity test must be highly specific and based on unique aspects of molecular structure, and treats identity, purity and content as separate determinations.
- Roepstorff and Fohlman fragment-ion nomenclature, Biomedical Mass Spectrometry, 1984. Origin of the a/b/c and x/y/z labelling used for the b and y fragment table in this article.
- USP general chapters on chromatography and on validation of compendial procedures. Basis for the system-suitability expectations cited: adequate retention clear of the void, resolution from the nearest impurity, and demonstrated specificity for an identity procedure.
- PubChem entry for the tetrapeptide Ala-Glu-Asp-Gly. Reference source for molecular formula, average and monoisotopic mass, and registry identifiers quoted in the parameter table.