Introduction: Why You Need the Right CRO for Unknown Impurity Identification
In the pharmaceutical lifecycle, encountering an unexpected impurity—especially during late-stage development—can be a critical roadblock. Whether it’s for regulatory compliance or ensuring patient safety, identifying and characterizing unknown impurities requires advanced analytical expertise and instrumentation. That’s where a specialized CRO for unknown impurity identification like ResolveMass Laboratories Inc. becomes indispensable.
In this blog, we walk you through a real-world case study based on our published research, “Identification, Isolation and Structure Elucidation of Novel Forced Degradation Products of Alectinib Hydrochloride,” where our team successfully identified four novel degradation impurities using LC-MS/MS, FTIR, NMR, and prep-HPLC—a full-spectrum example of our capabilities as a trusted CRO for unknown impurity identification.
Why Regulatory Agencies Demand Unknown Impurity Identification
If you’re in Phase 2 or Phase 3 drug development, Health Canada, FDA, and EMA require full identification of unknown impurities above qualification thresholds per ICH guidelines (Q3A, Q3B). When impurity peaks appear in stability or stress studies:
- You must isolate and structurally characterize them.
- You need a validated method to quantify them.
- You must explain their origin and toxicological impact.
This is exactly where partnering with a CRO for unknown impurity identification like ResolveMass ensures success, speed, and submission-readiness.
👉 Learn more about our custom synthesis capabilities and analytical services.
Case Study – Alectinib Degradation Products Under Oxidative Stress
Background:
- Drug: Alectinib Hydrochloride (used in ALK-positive NSCLC)
- Objective: Conduct a forced degradation study and identify all unknown impurities formed.
- Challenge: Unknown peaks detected only under oxidative stress, requiring full impurity profiling.
Our team at ResolveMass executed a complete analytical strategy to confirm the identification of four degradation products (DP-1 to DP-4) — all previously unreported in the literature.
CRO for Unknown Impurity Identification – Our Analytical Workflow
1. Forced Degradation Study (ICH Q1A Guidelines)
- Alectinib was subjected to:
- Acidic, basic, thermal, photolytic, and oxidative stress
- Result: No significant degradation except under oxidative conditions
- Outcome: Four unknown impurities (DP-1 to DP-4) detected
2. Isolation of Impurities via Prep-HPLC
- Preparative HPLC was used to isolate each degradation product
- Mobile phase: ammonium formate buffer and acetonitrile
- Flow rate: 25 mL/min with UV detection at 230 nm
3. LC-MS/MS for Molecular Mass Identification
- Instrument: Waters LC-MS/MS with positive-mode ESI
- Parent mass of Alectinib: m/z 483.32
- Unknown impurities observed:
- DP-1: m/z 499.25
- DP-2: m/z 499.19
- DP-3: m/z 499.26
- DP-4: m/z 501.29
4. NMR and FTIR for Structural Elucidation
- NMR: Bruker 400 MHz using 1H, 13C, DEPT-135, HSQC
- FTIR confirmed presence/absence of functional groups like CN and NH2
- Confirmed each impurity’s structure and functional groups
Final Structures Identified by ResolveMass – A CRO for Unknown Impurity Identification
DP-1 – N-oxide Impurity
- Morpholine N-oxide form of Alectinib
- Mass: m/z 499.15
- Fragment ions: 396.25, 381.26, 368.30
- Structure confirmed by NMR chemical shift changes in morpholine region
DP-2 – Epoxide Impurity
- Epoxide ring formation in benzo[b]carbazole core
- Mass: m/z 499.26
- Major fragments: 412.28, 384.33, 358.28
- Merged singlet peaks in methyl regions in 1H NMR
DP-3 – N-hydroxy Impurity
- Addition of hydroxyl group to nitrogen
- Mass: m/z 499.19
- Fragment at 395.24 indicating hydroxy loss
- Proton resonance shifts confirm structural reorientation
DP-4 – Amide Impurity
- CN group converted to CONH2
- Mass: m/z 501.6
- New 1H NMR signal at δ ~7.30 for NH2 group
- CN peak missing in FTIR spectra
These findings showcase the technical depth and multi-technique capabilities of ResolveMass as a CRO for unknown impurity identification.
H2: Why Choose ResolveMass as Your CRO for Unknown Impurity Identification?
✔ Peer-Reviewed Publications:
Our work on Alectinib impurity profiling is published and publicly available, demonstrating scientific authority.
✔ End-to-End Impurity Profiling:
From isolation to MS/MS to NMR, we offer complete structural elucidation.
✔ ICH & Regulatory Alignment:
All studies follow ICH Q1A, Q3A, Q3B, Q6A — ensuring submission-readiness.
✔ Small Molecule & Custom Conjugate Expertise:
We specialize in kinase inhibitors, oncology drugs, and difficult-to-profile APIs.
H2: Use Cases for a CRO for Unknown Impurity Identification
Are you facing:
- An unknown peak >0.1% in a stability study?
- A potential oxidative degradant during accelerated storage?
- An ambiguous impurity at a late-phase manufacturing scale?
Then you need a CRO for unknown impurity identification with the expertise and speed of ResolveMass.
👉 Explore custom synthesis services
👉 Learn more about our analytical lab solutions
Conclusion: CRO for Unknown Impurity Identification Done Right
Whether it’s for regulatory compliance, root-cause analysis, or synthesis troubleshooting, identifying unknown impurities requires both scientific rigor and analytical finesse. At ResolveMass Laboratories Inc., we combine peer-reviewed expertise with advanced instrumentation to deliver actionable impurity data — fast.
If you’re looking for a proven CRO for unknown impurity identification in Canada or the United States, partner with us and turn complex problems into clear solutions.
📞 Contact ResolveMass Today for Your Impurity Profiling Needs
ResolveMass Laboratories Inc.: Experience, Expertise, and Trust You Can Count On
ResolveMass Laboratories Inc. has established itself as a trusted name in the domain of nitrosamine testing services in United States and Canada. With over a decade of dedicated experience, we have completed hundreds of successful nitrosamine testing and risk assessment projects for both domestic and international clients. Our scientists PhD level scientists who has specialized experience in Mass Spectrometry and Nitrosamine Impurity chemistry.
We are one of the few Canadian CROs to offer a complete in-house nitrosamine testing solution—from risk assessment to confirmatory analysis, regulatory documentation, and expert consultation. We continually invest in cutting-edge technologies and method development, keeping pace with evolving regulations and industry demands. We also provide the custom synthesis service of rare nitrosamine Impurities which are not available anywhere.
Our clients trust us because we not only deliver accurate results but also help them understand and resolve complex impurity challenges. Choose ResolveMass Laboratories for your transparent nitrosamine testing services in United States and Canada—where precision meets reliability.
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Parmar R, Rajput S, Mohan A. Identification, isolation and structure elucidation of novel forced degradation products of alectinib hydrochloride. Sep Sci plus. 2021; 4: 174–184. https://doi.org/10.1002/sscp.202000104
