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In this application note, we demonstrate how the ConcentratIR2™ (figure 1) enables clear resolution of amide bands in model pharmaceutical peptides Ziconotide, Calcitonin, and Leuprolide Acetate at concentrations relevant to early-stage formulation and quality control. These peptides serve as structural and analytical analogs for GLP- 1-type drugs, helping to establish infrared spectroscopy workflows for assessing purity, stability, and folding in peptide-based therapeutics.
Ensuring the integrity and consistency of peptide therapeutics at low concentrations is a growing analytical challenge in pharmaceutical development, particularly in the context of GLP-1 drug formulation, where protein- based active ingredients may need to be monitored at the µg/mL level. Traditional transmission FTIR spectroscopy often lacks the sensitivity required at these low concentrations, with amide I and II bands, critical for confirming peptide structure, frequently obscured by noise or solvent background. The Harrick ConcentratIR2™, equipped with a 23-bounce silicon ATR crystal, addresses this limitation by significantly enhancing signal strength through multiple internal reflections. This allows for reliable detection of secondary structural markers, even in dilute aqueous samples.
A commercial FTIR spectrometer equipped with a liquid nitrogen-cooled MCT-A detector and the ConcentratIR2™ ATR accessory, featuring a 23-bounce extended Si crystal, was used. Samples included Ziconotide Intrathecal Infusions (25 µg/mL), Calcitonin Injection (400 units/2 mL), and Leuprolide Acetate Injection (14 mg/2.8 mL), alongside their respective placebo/buffer solutions. Only 10 µL of each sample was deposited onto the Si ATR element.
Spectra were collected from 4000–400 cm ¹ at 4 cm ¹ resolution, averaging 128 scans (approximately 77 seconds per spectrum). Background spectra were acquired using the bare Si crystal. Placebo/buffer spectra were subtracted using spectral software to isolate protein-specific amide I (1650 cm ¹) and amide II (1550 cm ¹) bands, focusing on the 1800–1400 cm ¹ range for analysis.
Sample spectra with the placebo/buffer subtraction performed are shown in Figure 2. The Concentrate R2 delivered high-quality spectra with excellent signal-to-noise ratios, resolving amide 1 and amide il bands for all samples post subtraction. Ziconotide (25 µg/mL) showed distinct peaks at 1650 cm and 1550 cm with intensibes half that of Leuprolide Acetate (14 mg/2 8 ml, reflecting concentration differences on a common seale.
Figure 1: The Harrick ConcentratIR2™ 23-bounce ATR accessory.
Leuprolide Acetate also exhibited distinct amide bands. Calcitonin (200 units/ml) displayed weaker but resolvable amide-l and Il bands, despite its lower effective concentration, demonstrating the accessory’s sensitivity Spectral subtraction effectively removed buffer/water contributions, isolating protein-specific features.
Overlaid subtraction results confirmed unique protein signatures, with no overlap in baseline noise, affirming the ConcentratiR2 s precision for trace analysis. Diear resolution of the amide land bands is essential as it enables further processing of the spectral data to extract detailed information about the protein’s secondary structure, such as alpha-helical and beta-sheet content which are key indicators of peptide integrity, stability, and bioactivity.
The Concentrator, with its 23-bounce Si ATR crystal can successfully detect pharmaceutical proteins at low concentrations lug/ml. to mg/mL) when coupled with an MCT-Adetector, malingit an ideal QC tool for biopharmaceuticals Spectral subtraction isolates amide / and bands, revealing molecular details entical for drug identity and stability, its high sensitivity, minimal sample volume (10 µLJ. and rapid acquisition 177 seconds) streamline analysis. For industries requiring trace level protein validation, the ConcentratiR2 is a versatile accessory
Hiten Techno Products Corporation 912, The Capital, Adjoining Jio Garden, G Block,
Bandra Kurla Complex,Mumbai – 400051.
Tel: +91-22-66268000 (100 lines)
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