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Methods

Reversed-phase HPLC with UV detection for purity, electrospray LC–MS for identity, and the three add-on tests. Every parameter and every acceptance criterion is published so that a chemist can judge whether the method is fit for the analyte.

The method, in full

Column, gradient, flow, wavelength, source voltage, the identity criterion in parts per million. Published so that a chemist can judge whether it is fit for the analyte they are sending.

1 Vial as received logged, photographed 2 Dissolve whole by mass, 2.0 mg/mL 3 RP-HPLC C18 2.1 × 100 mm 4 UV 214 nm area percent 5 ESI+ MS identity, ppm 6 Report hash, accession
  1. Vial as received logged, photographed
  2. Dissolve whole by mass, 2.0 mg/mL
  3. RP-HPLC C18 2.1 × 100 mm
  4. UV 214 nm area percent
  5. ESI+ MS identity, ppm
  6. Report hash, accession
ColumnC18, 2.1 × 100 mm, 1.7 µm
Mobile phase AWater, 0.1% trifluoroacetic acid
Mobile phase BAcetonitrile, 0.1% trifluoroacetic acid
Gradient5–60% B over 20 min; where co-elution is suspected, a 14-percentage-point window over 40 min centred on the composition at which the analyte eluted in the first run (for example 18–32% B for a 1 kDa peptide, 45–59% B for semaglutide)
Flow0.30 mL/min
Column temperature40 °C
Injection2 µL
Sample preparationMass per vial: three vials, each vented, weighed and dissolved whole, by mass, in LAL reagent water (10 mM Tris of LAL grade for semaglutide, tirzepatide and retatrutide, the Tris stock added to mobile phase A with mixing) to 2.0 mg/mL nominal, the volume being the nominal fill in mg divided by 2.0 (a 5 mg fill in 2.50 mL), then the stock 1:10 in mobile phase A, 0.20 mg/mL; the masses and volumes are recorded and printed on every certificate. Purity and identity: the stock 1 + 1 in mobile phase A, 1.0 mg/mL, 2 µL, so 2 µg on column; identity from the purity solution 1 + 9. Endotoxin: an aliquot of that stock first, 1:40 in LAL reagent water, 0.05 mg/mL in the well. Water: two whole vials by oven Karl Fischer at the validated temperature
DetectionUV 214 nm for purity and content; 280 nm as a secondary channel, to flag aromatic-containing impurities (epitalon, GHK, BPC-157 and TB-500 have no chromophore there); reference wavelength off
IonisationESI+, 3.5 kV
Identity criterion[M+nH]n+ within 10 ppm of theoretical
Replicatesn = 3 injections of one preparation; their RSD is injection repeatability. Mass per vial uses three independent vials
Reporting threshold0.05% area (ICH Q3A(R2)), meaningful once the analyte’s measured limit of quantitation is at or below it; until then peaks are listed to 0.05% and no limit of quantitation is claimed

These are the conditions every analyte starts from. Validation fixes the per-analyte gradient, temperature and load (a polar tripeptide needs a lower starting composition than a lipidated 4 kDa peptide, which is usually run warmer), and the conditions printed on each certificate are the ones that sample was run under.

Method parameters, as published.

Identity by LC–MS

Electrospray, positive mode, 3.5 kV, scan range m/z 300–2200. The ions are acquired across the main HPLC peak, and the certificate ties their retention time to it within 0.2 min. The monoisotopic peak of each resolved isotope cluster is compared with the theoretical m/z computed from the formula; every recorded charge state must be within 10 ppm. A 10 ppm criterion needs isotopes resolved to charge 5+, so it is applied only on an instrument with a resolving power of 10 000 or better at m/z 1000, and the certificate names the instrument class and the resolving power; on a unit-resolution instrument (a quadrupole or an ion trap) no criterion tighter than 50 ppm is accepted. The MS run takes the purity preparation in its own mobile phase, 0.1% trifluoroacetic acid, with the ion suppression that implies; it is not re-made in formic acid.

System suitability, every run

  1. Blank: no interfering peak at the analyte’s retention time. A peak present in the blank is listed with its share of the area and excluded from the normalisation.
  2. Repeatability: five replicate standard injections, area RSD not more than 2% (USP <621>).
  3. Symmetry factor 0.8 to 1.8, measured at 5% of peak height.
  4. Resolution of the critical pair at least 1.5, from peak widths at half height.
  5. Plate number reported as apparent, since the run is a gradient; no criterion is applied to it.
  6. One integration policy: integration from 0.5 min in every injection, the same rules for every peak.
  7. Peaks below the reporting threshold stay in the total area and are not listed as impurities, so the area percents sum to 100 by construction.

If the critical pair is resolved below 1.5, the purity is printed as an upper bound (at most) and the open-point row says why.

The add-on tests

Mass per vial, water content and endotoxin, each as the model that composes the certificate enforces it.

Mass per vial

RP-HPLC–UV at 214 nm against an external standard: a four-level calibration of the reference standard, concentrations corrected for the standard’s stated content, r² at least 0.999, and the sample’s response inside the calibrated range. Three independent vials, each reconstituted whole and diluted, reported as mg per vial with the basis stated: free peptide when the standard’s content is its net peptide content. Where water was also run on whole vials, the certificate prints a mass balance: peptide, water, impurities, the counter-ion at its stoichiometric maximum, and what is left unassigned.

Water content

Coulometric Karl Fischer, USP <921> Method Ic; Ph. Eur. 2.5.32. Each replicate is the contents of one whole vial extracted into anhydrous methanol; the solvent blank and the drift (at most 20 µg/min) are subtracted, and a certified water standard is run with every batch, recovery 97.5–102.5% (laboratory criteria). Reported as % w/w of the powder, the mean and RSD of two vials.

Bacterial endotoxin

Kinetic chromogenic LAL, USP <85>; Ph. Eur. 2.6.14 Method D. The sample is reconstituted at 2 mg/mL and tested at 1:40, so the reporting limit is 0.1 EU/mg. A positive product control spiked mid-curve must recover 50–200% of its spike, which rules out inhibition and enhancement, and a negative control must show no onset. Reported as EU per mg of powder to two significant figures. No endotoxin limit is applied unless you supply one; for a research material none is published.

Reference standards

For most research peptides no pharmacopoeial reference standard exists, and the standard is a commercially sourced peptide whose net peptide content is assigned by its supplier, usually by amino-acid analysis; the mass-per-vial result is traceable to that assignment and to nothing more authoritative. The certificate names the standard, its supplier, catalogue number, lot, stated content and how the content was assigned, and the retention-time match uses the same material.

Uncertainty

Every quantitative result carries an expanded uncertainty (k = 2) combining the spread of the replicates with a method floor: ±0.3% area for purity, ±2% relative for content, ±0.1% w/w for water, ×/÷ 1.5 for endotoxin. Until a method’s validation has measured intermediate precision the floor is our prior estimate, the certificate labels it provisional, and the decision rule against a specification uses it as stated: a result is said to meet a limit with the uncertainty included only when the whole interval lies inside it.

Standard operating procedures

The procedure behind each step, from receipt to release, as the laboratory will run it. Each takes effect when a named reviewer approves it; the control block on page 1 says whether it has.

Quality manual

Procedures

Software validation

Validation protocols

Bench worksheets

Safety

Each is composed on the certificate’s own paper, and every parameter and limit it quotes is read from the same data the reports are, so those cannot disagree with the report a procedure governs; the procedure text itself is written by hand, and the control block on page 1 says whether a reviewer has approved it. Selectable text, every font embedded, nothing encrypted.

Validation

What a signed validation report establishes for an analyte, and what marks its row of the scope table validated.

  1. Specificity: the analyte peak is resolved from its known impurities and from the blank, and what it was not resolved from is recorded.
  2. Linearity across the working range, with the correlation coefficient and the residual plot retained.
  3. Repeatability and intermediate precision, reported as relative standard deviation rather than as a single figure.
  4. Accuracy, as recovery of the reference standard at three levels, for mass per vial; the working range; and solution stability over the length of a sequence.
  5. Limit of detection and limit of quantitation, measured rather than inferred from a signal-to-noise rule of thumb alone.
  6. A signed report, dated, naming the analyst. That signature is what marks a method validated.
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