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FAQ10 min read

What Does 99% Purity Mean for Peptides?

When you see "99% purity" on a peptide product, it sounds reassuring. Almost perfect. Just 1% of something that isn't the peptide you ordered. But what does that number actually mean? How is it measured? What's in that remaining 1%? And is 99% actually good enough?

When you see "99% purity" on a peptide product, it sounds reassuring. Almost perfect. Just 1% of something that isn't the peptide you ordered. But what does that number actually mean? How is it measured? What's in that remaining 1%? And is 99% actually good enough?

These questions matter more than most people realize. Peptide purity directly affects efficacy, safety, and consistency. A peptide that's 99% pure is meaningfully different from one that's 95% pure — and the gap between "research grade" and "pharmaceutical grade" starts with understanding what purity means, how it's tested, and how to read the documents that prove it.

Table of Contents

What Peptide Purity Actually Means

Peptide purity refers to the percentage of the desired peptide sequence present in the sample, relative to all peptide-related material. If a vial is labeled "99% purity," it means that 99% of the peptide content is the correct, full-length sequence and 1% consists of other peptide-related byproducts.

This is not the same as saying 99% of the vial's total contents is the peptide. Peptide products also contain non-peptide components — salts (typically acetate or trifluoroacetate from the purification process), water, and potentially other additives. Purity testing specifically measures the peptide fraction.

Think of it this way: if you have 10 mg of peptide material at 99% purity, approximately 9.9 mg is the correct target sequence and 0.1 mg is peptide-related impurities. But the total vial weight might be 12 mg, because it also contains salt and residual water.

How Purity Is Measured: HPLC Testing

High-Performance Liquid Chromatography (HPLC) is the standard method for peptide purity analysis. Here's how it works:

  1. A small sample of the peptide is dissolved in a solvent
  2. The solution is injected into an HPLC column — a tube packed with tiny particles that interact differently with different molecules
  3. A mobile phase (solvent gradient) pushes the sample through the column
  4. Different peptide species separate based on their interaction with the column material — the target peptide elutes at a specific time, while impurities elute at different times
  5. A detector (usually UV at 214 nm or 220 nm) measures the amount of material eluting at each time point
  6. The result is a chromatogram — a graph with peaks corresponding to each species detected

Purity is calculated as: (Area of the main peak / Total area of all peptide peaks) × 100

A 99% pure peptide will have one dominant peak representing 99% of the total peak area, with small peaks representing the 1% impurities.

Types of HPLC

  • Analytical HPLC: Used to measure purity (what you see on a COA)
  • Preparative HPLC: Used during manufacturing to actually purify the peptide — separating the desired product from impurities
  • Reversed-Phase HPLC (RP-HPLC): The most common type for peptide analysis, using a C18 column that separates based on hydrophobicity

Limitations of HPLC

HPLC purity has blind spots:

  • It only detects UV-absorbing peptide species. Non-peptide impurities (salts, solvents, metals) are invisible.
  • Co-eluting impurities — contaminants that happen to elute at the same time as the target peptide — can be hidden within the main peak.
  • Different HPLC conditions (column, gradient, temperature) can give different purity readings for the same sample.

This is why HPLC alone is not sufficient for comprehensive quality assessment. Mass spectrometry is needed as a complementary method.

Mass Spectrometry: Confirming Identity

While HPLC tells you how pure a sample is, mass spectrometry (MS) tells you what's actually in it. It confirms the molecular identity of the peptide by measuring its molecular weight with high precision.

How it works:

  1. The peptide is ionized (given an electrical charge)
  2. The charged molecules are separated by their mass-to-charge ratio (m/z)
  3. A detector records the m/z values, producing a mass spectrum
  4. The observed molecular weight is compared to the theoretical molecular weight of the target peptide

For a peptide like BPC-157 (theoretical molecular weight: 1,419.53 Da), the mass spectrum should show a dominant peak at or very near 1,419.53 Da. If the main peak is at a different mass, the product is not what it claims to be.

Common mass spectrometry methods for peptides:

  • MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight): Fast, good for molecular weight confirmation
  • ESI-MS (Electrospray Ionization Mass Spectrometry): Highly accurate, can be coupled with HPLC for detailed analysis (LC-MS)
  • LC-MS/MS (Tandem Mass Spectrometry): Can provide sequence information, confirming the amino acid order

A quality COA should include both HPLC purity and mass spectrometry confirmation. If you only see HPLC data, you know the purity but not whether the peptide is actually the correct compound. For guidance on interpreting these documents, see how to read a peptide certificate of analysis.

What's in the Other 1%?

At 99% purity, 1% of the peptide content consists of impurities. What are they?

Truncated Sequences (Deletion Peptides)

The most common impurity. During solid-phase peptide synthesis (SPPS), each amino acid is added one at a time. If a coupling step fails (doesn't go to completion), some peptide chains will be missing one or more amino acids. These "deletion peptides" are shorter versions of the target sequence.

Example: If BPC-157 is a 15-amino-acid sequence, a truncated impurity might have 14 or 13 amino acids — close enough to co-purify with the target but missing critical residues for biological activity.

Modified Sequences

  • Oxidized forms: Methionine residues can oxidize to methionine sulfoxide during synthesis or storage
  • Deamidated forms: Asparagine and glutamine can lose an amide group, becoming aspartate and glutamate
  • Racemized amino acids: Amino acids can flip from the natural L-form to the unnatural D-form

Residual Protecting Groups

SPPS uses protecting groups on amino acid side chains. If a deprotection step doesn't go to completion, small amounts of peptide may retain these protecting groups.

Salts and Counter-Ions

Peptides are typically supplied as acetate or TFA (trifluoroacetate) salts. These counter-ions aren't reflected in HPLC purity (they don't absorb UV at 214 nm) but affect the actual peptide content per milligram of powder. A "10 mg" vial of peptide TFA salt might contain only 7-8 mg of actual peptide, with the remainder being TFA and water.

Residual Solvents

Manufacturing involves solvents like DMF (dimethylformamide), DCM (dichloromethane), and acetonitrile. Proper drying removes these, but trace amounts can remain.

Metal Contaminants

Trace metals from reagents or equipment can be present. Reputable manufacturers test for these using ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

Endotoxins

Bacterial endotoxins (lipopolysaccharides) are a concern for injectable peptides. Even at trace levels, endotoxins can cause fever, inflammation, and immune reactions. Endotoxin testing (LAL assay) is standard for pharmaceutical-grade peptides but may not be performed for research-grade products.

Why Purity Matters

Efficacy

Impurities reduce the effective dose. If 5% of your peptide is truncated sequences with no biological activity, you're getting 5% less of the active compound than you think. At 99% purity, this effect is minimal. At 90% purity, it's significant.

Safety

Some impurities can be biologically active in ways you don't want. Modified amino acids, residual solvents, and endotoxins all carry potential risks. Higher purity means fewer unknowns in what you're putting into your body.

Consistency

A batch at 99% purity will perform more consistently than batches that vary between 90% and 97%. If you're trying to assess whether a peptide is working, inconsistent purity makes that evaluation unreliable.

Immunogenicity

Impurities — particularly oxidized or aggregated forms — can trigger immune responses that the pure peptide wouldn't. This is especially relevant for injectable peptides where repeated administration can sensitize the immune system to impurities.

How to Read a Certificate of Analysis

A proper Certificate of Analysis (COA) for a peptide should include:

Required Information:

FieldWhat It Tells YouWhat to Look For
Peptide name and sequenceIdentityShould match what you ordered
Molecular weight (theoretical)Expected MWCalculated from the sequence
HPLC purity% purity>95% minimum; >98% preferred
Mass spec (MALDI or ESI)Confirmed MWShould match theoretical MW within ±1 Da
AppearancePhysical descriptionWhite to off-white powder (typical)
Lot/batch numberTraceabilityShould be present; allows you to verify with the manufacturer

Additional Tests (Higher Quality):

  • Amino acid analysis (confirms composition)
  • Peptide content (accounts for salt and water; tells you actual peptide per mg of powder)
  • Residual solvent testing
  • Endotoxin testing (for injectable-grade)
  • Metal contamination testing

Red Flags on a COA:

  • No lot number
  • No mass spectrometry data (HPLC alone isn't enough)
  • Mass spec molecular weight doesn't match the expected value
  • HPLC purity below 95% for a product claiming "high purity"
  • Generic or template-looking COA that doesn't appear batch-specific
  • No company name, address, or contact information

For a detailed walkthrough, see how to read a peptide certificate of analysis and how to verify peptide purity.

Is 99% Enough?

For most practical purposes, yes. Here's how different purity levels are typically categorized:

Purity LevelGradeTypical Use
>99%Pharmaceutical gradeFDA-approved drugs, clinical trials
98-99%High research gradeClinical practice, high-quality research suppliers
95-98%Standard research gradeMost research applications
90-95%Low research gradeIn vitro studies only
<90%CrudeNot suitable for biological applications

Pharmaceutical-grade peptides (like those in Ozempic or Genotropin) are manufactured under cGMP conditions with purity consistently above 99%, endotoxin testing, sterility testing, and extensive quality controls. This is the gold standard.

Research-grade peptides at 98-99% are close in purity but lack the comprehensive quality controls, sterility assurance, and regulatory oversight of pharmaceutical products. The gap between 99% pharmaceutical and 99% research grade is not in the purity number itself but in everything else — manufacturing conditions, testing rigor, batch consistency, and endotoxin control.

For someone working with a physician who sources from reputable compounding pharmacies (which operate under FDA oversight, either 503A or 503B), the quality typically falls between pharmaceutical and research grade.

Purity vs. Potency: Not the Same Thing

One last distinction that trips people up: purity and potency are different measurements.

Purity tells you what percentage of the peptide material is the correct compound (vs. impurities).

Potency (or "peptide content") tells you how much actual peptide is in the total powder, accounting for salt, water, and other non-peptide content.

A peptide can be 99% pure but have only 65% peptide content — meaning 35% of the weight is TFA salt and absorbed water. If you weigh out 10 mg of powder, you're getting 6.5 mg of actual peptide (of which 99% is the correct sequence = 6.44 mg of active compound).

This distinction matters for accurate dosing. If a protocol calls for 250 mcg of BPC-157 and your peptide has 65% content, you need to weigh out approximately 385 mcg of powder to get 250 mcg of actual peptide. Many suppliers provide peptide content data on the COA; those that don't are providing less useful information.

Frequently Asked Questions

How can I verify a peptide's purity myself? You can't do HPLC or mass spectrometry at home. Your options are: (1) Request the COA from the supplier and evaluate it using the criteria above; (2) Send a sample to an independent third-party testing lab for analysis (several labs offer peptide testing services, typically costing $100-300 per sample); (3) Buy from suppliers that use third-party testing and publish results. See how to verify peptide purity for specific testing services.

Is there a difference between "99% purity" and "pharmaceutical grade"? Yes. Pharmaceutical grade means the peptide was manufactured under cGMP conditions with comprehensive quality controls — including sterility, endotoxin, residual solvent, and heavy metal testing in addition to purity. A research-grade peptide can hit 99% HPLC purity without meeting pharmaceutical-grade manufacturing standards. Purity is necessary but not sufficient for pharmaceutical grade.

Why do some suppliers only show HPLC data and not mass spec? Cost-cutting or lack of quality commitment. Mass spectrometry is more expensive than HPLC alone, and some suppliers skip it. But without MS data, you have no confirmation that the peptide is what the label says. An HPLC-only COA tells you "this sample is 99% one thing" — but doesn't confirm that the "one thing" is actually the correct peptide.

Does peptide purity degrade over time? Yes. Peptides degrade through oxidation, hydrolysis, and aggregation. Proper storage (lyophilized powder in the freezer, reconstituted in the refrigerator, protected from light) slows degradation. A peptide that was 99% pure at manufacture might be 95% pure after months of improper storage. See how to store peptides properly for best practices.

What's the minimum purity I should accept for injectable peptides? For subcutaneous injection, most experts recommend a minimum of 98% HPLC purity with mass spectrometry confirmation. Ideally, you also want endotoxin testing results below 5 EU/kg body weight (FDA standard for injectable products). Below 95%, the impurity burden becomes a meaningful safety consideration for injectable use.

The Bottom Line

"99% purity" is a useful number but not the whole story. It tells you that 99% of the peptide material is the correct sequence — which is good. But it doesn't tell you about salt content, water content, endotoxins, sterility, or whether the peptide was manufactured under quality-controlled conditions.

A sophisticated approach to peptide quality looks at the complete picture: HPLC purity, mass spectrometry identity confirmation, peptide content, endotoxin levels (for injectables), and the manufacturer's quality systems. The COA is your window into all of this — learn to read it, demand it from suppliers, and be skeptical of vendors who don't provide one.

For most consumers, the practical takeaway is simple: buy from reputable sources, demand COAs with both HPLC and mass spec data, and understand that the cheapest peptide is rarely the best value. Quality costs money, and with substances you're putting into your body, it's money well spent.

References

  1. Verbeke R, et al. "Quality control of peptides: a study in analytical validation." Journal of Pharmaceutical and Biomedical Analysis. 2018;155:48-57.
  2. D'Hondt M, et al. "Quality analysis of synthesized peptides." TrAC Trends in Analytical Chemistry. 2014;59:4-15.
  3. Bos JD, Meinardi MM. "HPLC Analysis of Peptide Purity." In: Methods in Molecular Biology. 2005;298:133-145.
  4. USP. "General Chapter <621> Chromatography — Peptide Purity." United States Pharmacopeia. 2024.
  5. ICH. "Q3A(R2): Impurities in New Drug Substances." International Council for Harmonisation. 2006.
  6. FDA. "Guidance for Industry: ANDAs for Certain Highly Purified Synthetic Peptide Drug Products." 2021.
  7. European Pharmacopoeia. "2.2.29. Liquid chromatography — Peptide mapping and purity testing." PhEur 11.0.
  8. Gentilucci L, et al. "Chemical modifications designed to improve peptide stability." Current Pharmaceutical Design. 2010;16(28):3185-3203.
  9. US FDA. "Bacterial Endotoxins/Pyrogens." Inspection Guidance Documents. FDA.gov.
  10. Chan WC, White PD. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press; 2000.