Impurity profiling of the most frequently encountered falsified polypeptide drugs on the Belgian market

Summary

This is a comprehensive impurity-profiling study of falsified injectable research peptides, and for the Heavy Metal Index it is the highest-toxicological-significance occurrence finding in the June 8 research-chemical set. The authors acquired 27 unregulated polypeptide preparations sold as “research chemicals” through three illegal internet pharmacies and measured elemental impurities by ICP-MS against the ICH Q3D parenteral limits. Six of the preparations exceeded the class-1 arsenic limit of 1500 ppb, ranging from 1660 to 12,890 ppb — more than ten times the allowed concentration — and a follow-up HPLC-ICP-MS speciation confirmed that the arsenic was present entirely as inorganic arsenic, the most toxic and carcinogenic form. One preparation exceeded the lead limit, and single-sample exceedances were also found for copper, silver, and barium. Because these products are self-injected, an inorganic-arsenic load at ten times the parenteral limit is a serious, previously-undocumented exposure route. The finding establishes arsenic (specifically inorganic arsenic) as the priority elemental contaminant for the unregulated injectable-peptide category.

Key numbers

ICP-MS elemental impurities versus ICH Q3D parenteral limits. Concentrations in ppb (µg/kg); 1500 ppb = 1.5 mg/kg.

• The decisive finding: arsenic speciation (HPLC-ICP-MS) confirmed that all the elevated arsenic was inorganic arsenic (iAs), not the less-toxic organic forms.
• Maximum arsenic 12,890 ppb (≈12.9 mg/kg), roughly 8.6× the 1500 ppb ICH parenteral limit; the lowest exceeding sample (1660 ppb) was just over the limit.
• Silver and barium also showed exceedances; no platinum-group metal-catalyst residues were detected.
• Residual solvents were present in every sample (20–900 ppb) but below ICH Q3C limits; small-molecule contaminants (mannitol, sorbitol, PEG, residual reagents) were common.

Methods (brief)

Elemental impurity analysis used three platforms: an Agilent 8800 triple-quadrupole ICP-MS (octopole collision/reaction cell, He as collision gas, O₂ in the octopole for arsenic (As) and Se) for most elements; a Varian 820 ICP-MS with H₂ collision gas for lead (to remove interferences); and an Advanced Mercury Analyzer AMA 245 (PS Analytical) for mercury on 100 µL of digest. Arsenic speciation was carried out on a second batch by HPLC-ICP-MS (Varian, PRP-X-100 anion-exchange column, 40 mM ammonium carbonate eluent, pH 9.4), able to distinguish inorganic As(III)/As(V) from methylarsonate, dimethylarsinate and arsenobetaine. Sample prep: ≥2.0 mg of each peptide acid-digested (0.5 mL HNO₃ + 0.125 mL HCl + 0.25 mL H₂O₂, ramped to 90 °C and held 7 h), then diluted to 25 mL. Reference materials: NIST 1573a tomato leaves (trueness 88–98 % for cadmium (Cd), Co, Cu) for the elemental panel and NMIJ 7532a brown rice flour (inorganic As 0.298 ± 0.008 mg/kg) for the speciation run. API identity by LC-IT-MSⁿ on a Dionex Ultimate 3000 / Bruker amaZon system with tryptic digestion; small-molecule contaminants by GC-MS (Agilent 7890A / 5975C) and LC-MS² (Waters Synapt G2Si); residual solvents by HS-GC-MS. All elemental results benchmarked against ICH Q3D parenteral limits and solvents against ICH Q3C. Vanadium trueness was 130 % (interference not fully removable), so V results are not reliable; for mercury (Hg) and nickel (Ni) the per-sample material was below LOQ in many vials, so the absence of exceedances for those two elements is a detection-limit gap, not a clean result.

Implications

• Establishes inorganic arsenic as the priority elemental contaminant for Research Chemicals, Peptides & SARMs (injectable research peptides), based on speciation-confirmed iAs at 1660–12,890 ppb in 6 of 27 preparations.
• The iAs speciation result is the load-bearing toxicological point: it elevates the concern well beyond what total-arsenic screening alone would imply, and it routes to Arsenic as confirmed inorganic-As occurrence in a self-injected product.
• Supports a certification/screening case for elemental-impurity testing of unregulated injectable peptides against ICH Q3D parenteral limits.

Related evidence

• Lead

Update history

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