Hexarelin vs GHRP-6: Confirming a 14 Dalton Difference
Hexarelin vs GHRP-6 is a comparison that comes down to one carbon atom. The two hexapeptides share five of their six residues exactly, and at the sixth, hexarelin carries a methyl group on the indole ring of its D-tryptophan. That adds 14 daltons: trivial for a mass spectrometer, easy to lose on a steep HPLC gradient, and just ambiguous enough to cause real confusion when a lab is checking a shipment. This article explains where the difference sits, how to confirm it, and what to specify when ordering either compound in volume.
Where the methyl group sits
Laid side by side, the sequences are:
- GHRP-6: His-D-Trp-Ala-Trp-D-Phe-Lys-NH2
- Hexarelin: His-D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2
Only position 2 differs. In hexarelin, the D-tryptophan carries a methyl group at carbon 2 of the indole ring. It is a modified tryptophan rather than a different amino acid, which is part of the reason older listings sometimes treat the two compounds as near-synonyms. Both contain exactly two D-residues (position 2 and the D-phenylalanine at position 5), and both end in a C-terminal amide.
Hexarelin vs GHRP-6 by the numbers
| Property | GHRP-6 | Hexarelin |
|---|---|---|
| Formula | C46H56N12O6 | C47H58N12O6 |
| Monoisotopic mass | 872.44 Da | 886.46 Da |
| Average mass | 873.0 Da | 887.0 Da |
| [M+H]+ | m/z 873.45 | m/z 887.47 |
| [M+2H]2+ | m/z 437.23 | m/z 444.24 |
One CH2 unit, 14.016 Da, is the entire compositional difference. These are free-base masses. Both peptides are normally supplied as acetate or TFA salts, and the counter-ion adds to the weighed mass without appearing in the molecular ion.
Why a 14 Da gap is easy on MS and hard on HPLC
On the mass spectrometer
Any instrument used for peptide work separates m/z 873 from m/z 887 without difficulty, and the doubly charged ions are just as distinct. Intact mass will tell you which compound dominates a sample.
There is one trap. A +14 Da shift is also what an unwanted methylation looks like, and methylation can occur as a synthesis side reaction. In a mixed or poorly purified sample, a peak 14 Da above GHRP-6 could be hexarelin, or it could be a GHRP-6 molecule methylated somewhere else. Intact mass alone cannot tell them apart.
Fragmentation settles it
Tandem MS locates the extra mass. The C-terminal portion of both peptides, Ala-Trp-D-Phe-Lys-NH2, is identical, so the y-ion series should match between the two. The N-terminal b-ions tell the story: from b2 onward, the hexarelin series should run 14 Da heavier than GHRP-6, because the methyl sits on residue 2. If instead the shift appears only in the y-series, the extra mass is somewhere other than the indole ring and the sample is not hexarelin.
On the column
An aromatic methyl adds a little hydrophobicity, so hexarelin generally elutes slightly after GHRP-6 on a C18 column. The gap is small. A steep gradient can squeeze the two peaks together until they merge, and a merged pair reported as one peak overstates the purity of whichever compound the method was set up for.
When the goal is to separate close analogues rather than simply measure gross impurities, a shallow gradient is the better tool. Around one percent organic per minute or less will resolve pairs that a fast ramp will not. If you ever receive a certificate for either compound with a very fast gradient, it is fair to ask whether the method could have separated the other.
The unmodified tryptophan at position 4
Methylating one indole does not protect the other. Hexarelin still carries an ordinary tryptophan at position 4, the residue most easily oxidised while a peptide is being made, purified or stored. An oxidised species appears 16 Da above the parent, near m/z 903.5 for the singly protonated ion, and usually elutes a little earlier because it is more polar.
The methylated indole at position 2 is less reactive but not immune. A certificate reporting a +16 impurity usually does not say which tryptophan was oxidised, and working that out needs fragmentation data. For a lab tracking stability across a long-held order, the practical point is simpler: record the +16 level on arrival, and watch whether it grows.
Detection wavelength matters
With two indole rings and a phenylalanine, hexarelin absorbs well at 280 nm. That makes 280 nm tempting, but impurities that lack aromatic residues, such as some truncated sequences, are invisible there. At 214 nm every peptide bond contributes. The same lot can therefore show two different purity figures depending on wavelength, and a figure is only meaningful when the wavelength is stated with it.
Why the methyl group was added
The 2-position of the indole ring is a known chemical and metabolic weak point in tryptophan-containing peptides. Substituting it alters the ring’s electronics. In the structure-activity literature on this family, the change was reported to affect stability and binding at the growth hormone secretagogue receptor (GHSR-1a) relative to GHRP-6, and hexarelin has also been studied in vitro for binding to the scavenger receptor CD36. These are receptor-level findings from published research, not statements about any application.
What to put on a volume order
- The compound name and the full sequence, with the 2-methyl group on the D-tryptophan written out.
- The expected monoisotopic mass: 886.46 Da for hexarelin, 872.44 Da for GHRP-6.
- HPLC purity at a stated wavelength, ideally 214 nm, from a gradient shallow enough to resolve the 14 Da neighbour.
- The reported level of the +16 oxidised species.
- One lot for the whole order where possible, logged against every vial on receipt.
Bulk Peptides does not stock hexarelin or GHRP-6. Our growth hormone secretagogue range covers related research compounds, and the distinction between the secretagogue and GHRH-analogue families is set out in our comparison article.
Hexarelin and GHRP-6 are discussed here only as in-vitro research compounds and analytical subjects. Nothing in this article relates to human or veterinary use.
