Identity is confirmed by intact mass measurement, usually electrospray MS showing a coherent series of multiply charged ions that deconvolute to about 4493.3 Da average, read against a chromatographic retention time from a documented method. Purity percentage is a separate measurement and says nothing about which molecule eluted.
- LL-37 is the 37-residue mature cathelicidin peptide from the human hCAP18 precursor, sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES.
- Formula C205H340N60O53 gives an average mass of about 4493.3 Da and a monoisotopic mass of about 4490.58 Da.
- Electrospray ionisation of this strongly cationic peptide yields a 3+ to 7+ charge envelope, and a coherent multi-charge series is far stronger identity evidence than any single reported ion.
- Intact mass cannot distinguish the seven leucine and isoleucine positions, nor any permutation of the same residue set, so sequence confirmation requires fragmentation.
- HPLC purity is an area percentage that excludes counterion, water and salts; peptide content by amino acid analysis is the number that governs actual concentration.
- No approved medicine contains LL-37 and no compendial monograph or official reference standard exists for it.
Identity and purity answer different questions
A certificate of analysis that says Purity: 98.2% (HPLC) has told you that one peak accounted for 98.2 percent of the absorbance in a chromatogram. It has not told you what that peak was. Purity is a proportion; identity is an assignment. Conflating them is the single most common failure in how research-grade peptide documentation gets read, and it survives because the two numbers usually sit two lines apart on the same page.
The distinction has teeth with LL-37 specifically. It is a 37-residue synthetic target with eleven basic residues, seven leucine-or-isoleucine positions and no chromophore beyond phenylalanine. Several plausible synthesis failures produce material that runs as one clean peak at the expected retention time and is not LL-37. A deletion at one of the leucines shifts the mass by 113 Da and barely moves retention. A scrambled Leu/Ile assignment shifts the mass by nothing at all.
So the question a competent laboratory asks of an incoming lot is not how pure it is. It is: what evidence exists that this material is the sequence claimed, and does that evidence come from a method whose parameters are written down?
The molecule the certificate is claiming
LL-37 is the mature cathelicidin peptide released from the human hCAP18 precursor encoded by CAMP. UniProt entry P49913 records the 170-residue precursor; LL-37 corresponds to its C-terminal 37 residues. The name comes from the two leucines at the N-terminus and the length.
One-letter sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
Molecular formula C205H340N60O53, free acid at both termini, no modifications. Average mass about 4493.3 Da, monoisotopic mass about 4490.58 Da. Eleven basic side chains (six lysine, five arginine) against five acidic ones give a strongly cationic molecule with a calculated isoelectric point above 10.
Four absences matter more for analysis than most of what is present. There is no cysteine, so no disulfide bonding, scrambling or free-thiol assay to worry about. There is no methionine and no tryptophan, which removes the two residues that dominate oxidation profiles in most peptides. And there is no tryptophan or tyrosine at all, which means absorbance at 280 nm is essentially blind to this peptide. Any concentration figure on a certificate that appears to derive from an A280 reading should be queried, because the only UV signal available is the amide backbone in the 210 to 220 nm region and the four phenylalanines contributing weakly near 258 nm.
The peptide is unstructured in dilute aqueous buffer and adopts an amphipathic helix in membrane-mimetic or high-salt conditions. That conformational switch is well described in the antimicrobial peptide literature and it has a practical consequence: retention behaviour and apparent recovery both shift with buffer composition and concentration.
What the mass spectrum establishes
Electrospray ionisation of a peptide carrying eleven protonatable sites produces a distribution of charge states rather than a single ion. For LL-37 the useful window under standard acidic LC-MS conditions runs roughly from 3+ to 7+, with the envelope centre depending on source conditions and organic content at elution.
| Charge state | Calculated m/z (monoisotopic) | 13C isotope spacing (m/z) |
|---|---|---|
| [M+3H]3+ | 1497.87 | 0.33 |
| [M+4H]4+ | 1123.65 | 0.25 |
| [M+5H]5+ | 899.12 | 0.20 |
| [M+6H]6+ | 749.44 | 0.17 |
| [M+7H]7+ | 642.52 | 0.14 |
Two things make this table useful rather than decorative. First, the isotope spacing gives the charge state directly on any instrument that resolves it, so a claimed assignment can be checked against the raw spectrum without trusting the vendor software. Second, a coherent series is much stronger evidence than a single ion. If a certificate reports 4+ and 5+ and 6+ ions that all deconvolute to the same neutral mass within a few parts per million, an accidental match is unlikely. A single reported m/z with no charge state named is close to worthless.
Note that a low-resolution instrument reports the average-mass position instead of the monoisotopic one: on a quadrupole running unit resolution the 4+ ion appears near 1124.3, not 1123.65. Certificates that quote a mass to two decimal places from a method incapable of resolving isotopes are quoting a calculation, not a measurement. That is a small tell and it is a reliable one.
The other thing worth reading in a spectrum is what sits beside the main envelope. Sodium and potassium adducts (+21.98 and +37.96 on the neutral mass) are routine and mean nothing. Trifluoroacetate adducts appear where TFA is the counterion. Discrete additions of 56, 100 or 252 Da point at incompletely removed side-chain protecting groups from solid-phase synthesis, which is a real quality signal rather than an artefact.
Where intact mass runs out
Mass is degenerate. Several substitutions and rearrangements change nothing measurable, and a few change so little that only a high-resolution instrument sees them.
| Ambiguity | Mass difference | Relevance here |
|---|---|---|
| Leucine ↔ isoleucine | 0 Da | Seven positions affected; invisible to any mass measurement |
| Any permutation of the same residues | 0 Da | A scrambled coupling order gives an identical intact mass |
| Lysine ↔ glutamine | 0.0364 Da | About 8 ppm on 4.5 kDa; needs high resolution and good calibration |
| Asparagine ↔ aspartate (deamidation) | +0.984 Da | Asn30 and Gln22 are the candidate sites |
| Glutamate ↔ glutamine | 0.984 Da | Resolvable, but easily mistaken for deamidation |
Resolving these requires fragmentation. Top-down MS/MS on the intact peptide, or a limited enzymatic digest followed by peptide mapping, produces the b/y or c/z ion series that pins residues to positions. For a peptide this basic, trypsin is a poor choice because eleven K/R sites shred it into fragments too small to be informative; Glu-C or chymotrypsin give more tractable maps. Electron transfer dissociation tends to work better than collision-induced dissociation on highly charged precursors, which LL-37 readily forms.
Leucine and isoleucine remain unresolved even then, without specialised side-chain fragmentation. In practice most suppliers do not attempt it, and a laboratory that genuinely needs that level of assurance orders against a reference standard and compares.
Amino acid analysis after acid hydrolysis is the orthogonal composition check. It confirms residue ratios and, separately, delivers a number that intact mass never can: how much actual peptide is in the vial. USP General Chapter <1052> describes the method for biotechnology-derived articles.
Chromatography, read against the mass
The chromatogram earns its place in identity work by contributing retention behaviour, and it earns its place in purity work by separating things the mass spectrometer sees as one population. Both jobs depend on the method being written down. A purity figure without a column, a gradient, a mobile phase and a detection wavelength attached is an assertion.
LL-37 is awkward on reversed phase for a predictable reason. Cationic peptides interact with residual silanols, and the result is peak tailing that inflates apparent impurity or, worse, hides a co-eluting species under a broad front. Trifluoroacetic acid at around 0.1 percent as an ion-pairing agent gives much better peak shape than formic acid and suppresses ESI response in exchange. Elevated column temperature sharpens the peak. High-purity endcapped C18 or a charged-surface hybrid phase behaves better than older silica. None of this is exotic, but it means two laboratories running nominally similar methods can report meaningfully different purity numbers on the same lot.
Retention time alone identifies nothing. Co-injection against a characterised reference, or a second method operating on a different mechanism, is what makes chromatographic evidence carry weight. Cation exchange is genuinely orthogonal for a peptide with this charge, and a purity figure that holds across a reversed-phase and an ion-exchange method is a much stronger claim than 99 percent on one gradient. USP General Chapter <621> sets out the system suitability expectations that make either number defensible.
There is no compendial monograph for LL-37. It is not an approved drug substance, so no USP or Ph. Eur. reference standard defines what a conforming result looks like. Identity rests on the supplier's own characterisation and on whatever confirmation the receiving laboratory runs itself.
Purity percent, peptide content, and the arithmetic between them
HPLC purity is an area percentage measured at a wavelength where only peptide bonds absorb. Trifluoroacetate counterions, water, residual salts and solvent do not absorb at 214 nm and therefore do not appear in that number at all. A lot can be 97 percent pure by chromatography and well under 80 percent peptide by mass.
Take a documented example of the numbers a full certificate carries:
Nominal content: 5 mg | HPLC purity: 96.4% area at 220 nm | Peptide content (AAA): 78.2% | TFA: 8.9% | Water (Karl Fischer): 6.1%
Net peptide in the vial:
5 mg × 0.782 = 3.91 mg
Brought into a diluent volume of 1.00 mL, the nominal concentration is 5 mg/mL and the real one is 3.91 mg/mL. In molar terms, using the average mass:
3.91 mg ÷ 4493.3 g/mol = 0.870 µmol → 870 µM
Against a nominal 1113 µM. That is a 22 percent error carried silently into every downstream calculation, and it is invisible unless someone reads the peptide content line. The vial concentration calculator handles the same arithmetic for other vial sizes and volumes; the cost per mg tool is the place to notice that price comparisons between lots are meaningless until both are expressed per mg of net peptide.
Residual TFA has a second consequence beyond mass accounting. It is not inert in cell-based assays, and where a protocol is sensitive to it the counterion needs to be exchanged to acetate or hydrochloride, which is a specification to request at ordering rather than discover later.
Reading the certificate line by line
| Field | What it should say | If missing |
|---|---|---|
| Sequence | Full one-letter code, termini stated (free acid, free amine) | You cannot check the mass against anything |
| Molecular formula and theoretical mass | Formula plus both average and monoisotopic values | Usually copied from a catalogue, not calculated for this lot |
| Observed mass | Measured m/z with charge state named, and the deconvoluted neutral mass | "MS: conforms" is not identity evidence |
| MS method | Ionisation mode and instrument class (ESI-TOF, ESI-Q, orbital trap) | Reported decimal places cannot be assessed |
| HPLC method | Column, gradient, mobile phase modifier, wavelength, retention time | The purity number is not reproducible or comparable |
| Chromatogram | Actual trace with labelled axes and a visible baseline | Tailing and co-elution are hidden |
| Peptide content | Percentage with the method named (AAA or nitrogen determination) | Assume every concentration is overstated by an unknown margin |
| Counterion and water | Salt form, percentage, water by Karl Fischer or TGA | Mass balance cannot be closed |
| Lot, date, analyst | Unique lot identifier, analysis date, responsible laboratory | The document is not traceable to material |
Red flags, in rough order of how often they appear: a mass quoted to four decimal places from a unit-resolution instrument; a chromatogram image with the y-axis cropped away; purity above 99 percent with no trace attached; the identical chromatogram appearing under two different lot numbers; theoretical mass on the certificate that does not match the sequence printed above it. That last one takes thirty seconds to check with a formula calculator and it catches template errors more often than anyone would like.
Where the receiving laboratory has LC-MS access, confirming intact mass in-house on arrival is cheap insurance and creates a record independent of the supplier. Our quality standard page sets out which of these fields we treat as mandatory before a lot is released, and the bacteriostatic water guide covers the diluent-side documentation that pairs with it.
Regulatory position
LL-37 is an endogenous human host-defence peptide described extensively in innate immunity research. It is not the active ingredient of any medicine authorised by the FDA, the EMA or any comparable regulator, and there is consequently no compendial monograph, no official reference standard and no pharmacopoeial identity test to conform to. Material supplied for research is characterised against the supplier's own analytical package, which is why reading that package properly is the whole of the diligence available.
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 LL-37 studied for?
Published research on LL-37 investigates the areas below, which is a different question from what LL-37 will do for anyone, a claim about a living system that nothing on this site is sold for.
What it is. The 37-residue C-terminal fragment of human cathelicidin hCAP18 — an amphipathic helical host-defence peptide.
What the research looks at. Innate immunity and host defence, antimicrobial mechanism, inflammation, angiogenesis and wound healing. A large mainstream immunology literature.
How it is thought to work. Direct antimicrobial activity by membrane disruption, acting as a pore-forming peptide against bacterial membranes. Separately functions as an alarmin, modulating inflammation in both directions and promoting angiogenesis and wound repair. Its tendency to oligomerise in physiological solution is central to both.
What is not established. No approved product. LL-37 is cytotoxic to host cells as well as bacterial ones, and the therapeutic window between the two is the central unresolved problem.
The full record, including the certificate for the lot in stock, is on the LL-37 product page.
Common questions
What mass should an LL-37 certificate report?
Can mass spectrometry alone confirm the sequence?
Why is absorbance at 280 nm useless for this peptide?
How much does residual TFA matter?
Is there an official reference standard for LL-37?
More documentation guides
Sources
- UniProt entry P49913 (CAMP_HUMAN, cathelicidin antimicrobial peptide). Establishes the 170-residue hCAP18 precursor and the mature LL-37 sequence and length used throughout this article.
- USP General Chapter <621> Chromatography. Defines system suitability and method reporting requirements that make an HPLC purity figure reproducible and comparable between laboratories.
- USP General Chapter <1052> Biotechnology-Derived Articles — Amino Acid Analysis. Describes acid hydrolysis and amino acid analysis, the basis for composition confirmation and for peptide content determination as distinct from chromatographic purity.
- ICH Q6B, Specifications for Biotechnological and Biological Products. Supports the separation of identity, purity and content as independent specification categories requiring independent analytical evidence.
