--- title: "Peptide Testing Glossary" description: "Plain definitions of the terms on a peptide certificate of analysis: HPLC, net peptide content, endotoxin, mass spectrometry and more." url: https://www.certuspeptides.com/research/glossary markdown: https://www.certuspeptides.com/research/glossary.md site: Certus Peptides --- # Peptide Testing Glossary Definitions of the terms used on peptide certificates of analysis and across this site. ## HPLC (high-performance liquid chromatography) A separation technique in which a sample is pushed through a packed column under pressure so that its components leave the column at different times. For peptides, reversed-phase HPLC with UV detection at 214 nm is the standard purity method, separating species by hydrophobicity. ## Area percent purity The area of the main HPLC peak divided by the total area of all detected peaks, expressed as a percentage. It describes the relative proportion of UV-absorbing species in a sample and does not measure salt, water or peptide content. ## Net peptide content The fraction of a lyophilized cake's mass that is peptide, as opposed to counter-ions, water and residual solvent, usually measured by amino acid analysis. Typical values for a trifluoroacetate salt are 80-90 %, and it is the number that governs concentration calculations. ## Mass spectrometry An analytical method that ionizes a sample and measures the mass-to-charge ratio of the ions, from which molecular weight is determined. For peptides it confirms identity by comparing the observed mass with the mass calculated from the sequence, but it cannot distinguish isomers of equal mass. ## LC-MS (liquid chromatography-mass spectrometry) The coupling of an HPLC separation to a mass spectrometer so that each chromatographic peak is mass-analyzed as it elutes. Electrospray LC-MS is the standard identity method for synthetic peptides and also reveals the mass, and therefore the likely origin, of each impurity peak. ## MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, in which the sample is co-crystallized with a UV-absorbing matrix, ionized by a laser pulse and mass-analyzed by flight time. It produces mostly singly charged ions, which makes spectra simple to read, but it is less quantitative than LC-MS and is not coupled to a separation. ## Certificate of analysis (COA) A document issued for a specific lot listing each test performed, the method, the specification, the measured result and whether it passed, together with the laboratory, the test date and the lot number. A certificate is only evidence about a vial whose lot number matches it exactly. ## Lot number The identifier printed on a vial, its box and its certificate of analysis that links the three to one production and testing event. Certus lot numbers take the form CP-BPC157-2607A and resolve at /lab-reports/{lot}. ## ISO/IEC 17025 The international standard for the competence of testing and calibration laboratories. Accreditation to ISO/IEC 17025 means an independent accrediting body has verified the laboratory's methods, equipment, staff competence and quality system for the specific tests in its scope. ## Endotoxin Lipopolysaccharide from the outer membrane of Gram-negative bacteria, which persists after the bacteria are killed and activates innate immune signaling through TLR4 at very low concentrations. It is measured separately from sterility because a sterile solution can still contain endotoxin. ## EU/mg (endotoxin units per milligram) The unit in which endotoxin content of a peptide lot is reported, normalized to peptide mass so that the endotoxin load at any working concentration can be calculated. Certus releases lots at under 5.0 EU/mg, with typical measured values below 1.0. ## LAL test (Limulus amebocyte lysate) An assay for bacterial endotoxin based on the clotting reaction of horseshoe crab amebocyte lysate. Kinetic chromogenic and turbidimetric variants quantify endotoxin in endotoxin units, and the method is the pharmacopoeial standard for reporting EU/mg. ## Sterility The absence of viable microorganisms in a sample, demonstrated by incubating the material or a filtered portion of it in growth media and observing no growth. Sterility and endotoxin are independent tests answering different questions. ## Lyophilized Freeze-dried: frozen and then dried under vacuum so that water sublimes directly from the solid, leaving a porous cake. Lyophilization removes the water in which degradation reactions run and is the standard form in which synthetic peptides are supplied and stored. ## Reconstitution Dissolving a lyophilized peptide in a chosen diluent to produce a solution of known concentration. Diluent choice, gentle addition down the vial wall, no shaking, and aliquoting before freezing are the handling steps that preserve the material. ## Bacteriostatic water Sterile water containing 0.9 % benzyl alcohol as a preservative that inhibits bacterial growth in a multi-entry vial. It is unbuffered, is unsuitable for most cell culture work because of the preservative, and gives a stoppered vial roughly 28 days of protection under refrigeration. ## TFA salt (trifluoroacetate salt) The form in which most synthetic peptides are supplied, because trifluoroacetic acid is used in cleavage and HPLC purification and pairs with every protonatable site on the peptide when the material is dried. Trifluoroacetate is 114 Da per site and residual TFA is reported on the certificate. ## Acetate salt A peptide in which the trifluoroacetate counter-ions have been exchanged for acetate, 60 Da per site. Acetate salts have a higher net peptide content per milligram of cake and produce a less acidic solution, and the salt form should be stated on the certificate. ## Counter-ion The anion that balances the positive charge on a peptide's protonated amine groups in the solid state, most often trifluoroacetate or acetate. Counter-ions are the largest non-peptide contributor to cake mass and the main reason net peptide content is below 100 %. ## Molecular weight The mass of one molecule of a compound, for peptides usually quoted as the average mass calculated from the sequence using natural isotope abundances, in daltons. A certificate compares the LC-MS observed mass with this theoretical value, typically to a tolerance of 1.0 Da. ## Monoisotopic mass The mass of a molecule calculated using the most abundant isotope of each element, as opposed to the average mass. High-resolution mass spectrometry reports monoisotopic mass, and the two values diverge by several daltons for peptides above about 2,000 Da. ## Deamidation A degradation reaction in which an asparagine or glutamine side chain loses ammonia, converting to aspartate or isoaspartate (or glutamate) and adding 0.98 Da and a negative charge. Asparagine followed by glycine is the fastest motif, and the reaction requires water. ## Oxidation (peptide) The addition of oxygen to a susceptible residue, most commonly methionine to methionine sulfoxide, cysteine to a disulfide, or tryptophan to kynurenine and related products. Oxidized species are 16 Da heavier per oxygen and usually elute earlier on reversed-phase HPLC. ## Aspartimide A five-membered succinimide ring formed when an aspartate side chain cyclizes onto the adjacent backbone amide, most readily at Asp-Gly, Asp-Ser and Asp-Asn motifs. It reopens as a mixture of isomers with the same mass as the parent, so it is detected by a dedicated HPLC gradient rather than by mass spectrometry. ## Deletion sequence A synthesis impurity in which one residue failed to couple and the chain was completed without it, giving a peptide lighter than the target by that residue's mass. Deletion sequences elute close to the main peak on HPLC and are identified by LC-MS. ## Retention time The time from introduction of a sample onto an HPLC column to the elution of a peak under a specified method. It is only meaningful for identity when compared with a reference standard run under the same column, gradient and conditions. ## Reference standard A characterized sample of a compound of established identity and purity against which test samples are compared, by retention time, mass or chiral chromatography. It is the only way to confirm identity for impurities that mass spectrometry cannot see, such as diastereomers. ## GLP-1 receptor agonist A peptide that binds and activates the glucagon-like peptide-1 receptor, a class B G protein-coupled receptor signaling through Gs and cAMP. Semaglutide is a selective GLP-1 receptor agonist; tirzepatide and retatrutide engage GLP-1R alongside other receptors. ## GIP (glucose-dependent insulinotropic polypeptide) A 42-residue incretin hormone whose receptor, GIPR, is a class B G protein-coupled receptor. Tirzepatide is built on a GIP scaffold and is a full agonist at GIPR, and retatrutide engages GIPR as one of its three receptors. ## Ghrelin receptor (GHS-R1a) The growth hormone secretagogue receptor type 1a, a class A G protein-coupled receptor coupling through Gq, phospholipase C and calcium, whose endogenous ligand is acylated ghrelin. Ipamorelin and hexarelin are synthetic agonists at this receptor. ## GHRH analog A peptide derived from growth hormone releasing hormone that acts at the GHRH receptor, a class B G protein-coupled receptor signaling through Gs and cAMP. Sermorelin is the unmodified 1-29 fragment; modified GRF(1-29) and CJC-1295 carry stabilizing substitutions on the same frame. ## Secretagogue (growth hormone secretagogue) A synthetic ligand of the ghrelin receptor, GHS-R1a. The term covers the GHRP family, hexarelin and ipamorelin, and is distinct from GHRH analogs, which act at a different receptor through a different second-messenger pathway. ## DAC (drug affinity complex) A maleimidopropionyl group attached to a peptide, usually through a lysine side chain, that reacts covalently with the free thiol of cysteine 34 on serum albumin. On CJC-1295 it adds 279.3 Da, and its maleimide hydrolyzes on exposure to moisture, which is why maleimide integrity is assayed. ## PEGylation The covalent attachment of polyethylene glycol chains to a peptide to increase its hydrodynamic size and shield it from proteolysis and clearance. PEGylated peptides show a broad mass distribution on mass spectrometry because the polymer is polydisperse. ## Acylation (fatty acid acylation) The attachment of a fatty acid or fatty diacid, often through a linker, to a lysine side chain of a peptide so that the chain binds reversibly to serum albumin. Semaglutide carries a C18 diacid and tirzepatide a C20 diacid; the des-acyl form is a characteristic impurity. ## Cyclic peptide A peptide whose backbone or side chains are joined into a ring, by a lactam bridge, a disulfide bond or head-to-tail cyclization, constraining its conformation and resisting proteolysis. The uncyclized linear precursor differs by 18 Da and is a common impurity. ## Lactam bridge An amide bond formed between the side-chain carboxyl of an aspartate or glutamate and the side-chain amine of a lysine or ornithine, closing a ring within a peptide. Melanotan II and PT-141 are cyclized by an Asp-to-Lys lactam bridge. ## Disulfide bond A covalent bond between the sulfur atoms of two cysteine residues, forming a ring within a peptide or linking two chains. Cagrilintide and survodutide each contain a Cys7-Cys14 disulfide, and incorrect pairing or reduction of the bond is a quality defect detectable by mass and chromatography. ## Research use only (RUO) A regulatory designation for material intended for laboratory investigation and not for diagnostic use, human consumption or veterinary administration. RUO products have not been evaluated by the FDA, and no claim about diagnosis, treatment or outcomes may be attached to them. ## Cold chain The unbroken sequence of controlled-temperature storage and transport from the point of manufacture to the point of use. For lyophilized peptides this means storage at -20 °C, shipping with a cold pack and desiccant, and minimizing the number of temperature excursions. ## Quarantine lot A lot that is held and not shipped pending the completion of a further assay or an investigation. Certus holds retatrutide lots in quarantine for a second independent assay run before release, and the status is shown on the lot's certificate page. ## Retired lot A lot that has sold through or aged to the point where new orders are filled from a newer lot. A retired lot is not a failed lot; its certificate stays on file so that material already in the field can still be verified against it. ## Batch vs lot A batch is the quantity of material produced in one manufacturing run; a lot is the quantity released together under one identifier after testing. In peptide supply the terms are often used interchangeably, but the lot number, not the batch, is what a certificate of analysis refers to. ## Orthogonal methods Two or more analytical methods that measure different properties and fail in different ways, so that an error invisible to one is caught by the other. HPLC purity and LC-MS mass are the minimum orthogonal pair for peptide identity. ## Sequence (peptide) The ordered list of amino acid residues in a peptide from the N-terminus to the C-terminus, including any non-proteinogenic residues, D-amino acids, terminal modifications and bridges. The sequence defines the theoretical mass against which the observed mass is compared. ## Amino acid analysis The reference method for net peptide content: a weighed sample is acid-hydrolyzed to its constituent amino acids, which are quantified by chromatography against calibrated standards. The recovered amino acid mass divided by the sample mass gives the peptide fraction, and the residue ratios check composition. --- For laboratory and research use only. Not for human or veterinary consumption. Not a drug, food or cosmetic. Sold exclusively to qualified researchers and licensed laboratories. Support: support@certuspeptides.com. Every certificate of analysis: https://www.certuspeptides.com/lab-reports.