Is Your HPLC System Properly Calibrated? 5 Red Flags for Biopharma Labs
High-Performance Liquid Chromatography (HPLC) is a key piece in biopharmaceutical analysis, providing important data for everything from raw material qualification to final product validation. But even the most advanced HPLC systems are only as reliable as their calibration. In a regulated industry where data integrity, accuracy, and compliance are a requirement, overlooking calibration can jeopardize product quality, regulatory approval, and patient safety.
So how can your lab tell if an HPLC system is drifting out of spec? GL Technologies explores five common red flags that signal your HPLC system may not be properly calibrated. and what to do about them.
Why HPLC Calibration Matters in Biopharma
In biopharma and pharmaceutical environments, HPLC systems are used for analyzing APIs, impurities, stability samples, and excipients. Calibration ensures the system provides accurate, reproducible results, a necessity for meeting Good Manufacturing Practice (GMP) guidelines and FDA/EMA regulatory expectations.
A properly calibrated HPLC system guarantees:
Accurate retention times
Reliable peak quantification
Consistent resolution between analytes
Data traceability and integrity
Compliance with SOPs and regulatory audits
Skipping calibration, or doing it poorly, risks invalidating critical data, a costly mistake in an industry where each batch may represent millions of dollars in value.
Red Flag #1: Inconsistent Retention Times
One of the first signs of a miscalibrated HPLC system is inconsistent retention times. When the same compound starts eluting at different times across runs or instruments, calibration (particularly of the flow rate or gradient system). may be off.
Potential Causes:
Inaccurate pump flow rates due to wear or improper calibration
Fluctuations in column temperature
Failing gradient proportioning valves
Contamination or aging of the mobile phase
Why It Matters
Regulatory agencies expect retention times to remain within a validated window. Fluctuations can cause incorrect peak identification, leading to flawed impurity profiling or potency determination which can result in batch rejection or rework.
Solution
Use calibrated flowmeters and regularly verify pump performance. Make sure the system’s dwell volume and gradient formation are within expected limits. Include retention time standards in system suitability tests (SSTs) before each batch of analyses.
Red Flag #2: Poor Peak Shape or Resolution
Peaks that appear tailing, fronting, or overly broad may suggest that your HPLC system isn’t performing optimally, and calibration issues could be the issue.
Common Culprits:
Detector calibration drift
Incorrect injection volume settings
Contaminated or worn-out columns
Autosampler misalignment
Inaccurate wavelength settings for UV detectors
Why It Matters
Poor peak shape affects quantification accuracy and can obscure closely eluting peaks, such as process-related impurities or degradation products. In regulatory submissions, such issues could raise red flags and trigger requests for revalidation or additional data.
Solution
Verify detector linearity and wavelength calibration using traceable standards. Confirm injector precision by checking replicate injections. Replace or clean injector needles and check for column health routinely.
Red Flag #3: Failing System Suitability Tests (SST)
System Suitability Tests are required before sample runs to verify that the HPLC system is operating within validated performance specifications. Repeated SST failures often indicate deeper calibration or maintenance issues.
Indicators Include
Resolution between peaks below required thresholds
Tailing factors outside acceptable ranges
%RSD (relative standard deviation) of injections exceeding limits
Retention time shifts compared to historical data
Why It Matters
An SST failure invalidates the entire run. In high-throughput biopharma labs, this can result in significant delays, increased cost, and backlog, especially when working with stability samples on a timeline.
Solution
Keep a log of SST performance trends to identify when issues start. If SST parameters consistently fall short, recalibrate the relevant components (e.g., detectors, injectors, pump). Preventative maintenance and method revalidation may also be necessary.
Red Flag #4: Calibration Standards Deviating from Expected Results
Another red flag is when calibration standards, which are run to create a calibration curve, produce inconsistent or incorrect values. For example, linearity may drop, or the slope of the calibration curve may change unexpectedly.
Possible Issues
Detector drift or wavelength misalignment
Degraded reference standards
Pipetting errors during dilution
Autosampler volume inconsistencies
Temperature variations affecting analyte response
Why It Matters
Calibration curves are fundamental to accurate quantitation. Errors here can propagate across every sample analyzed using that curve. Regulatory audits often scrutinize these curves, and any deviation must be documented and justified.
Solution
Always use freshly prepared, validated calibration standards. Ensure proper storage conditions for standards and use calibrated pipettes. Revalidate the detector’s response curve at required intervals and log all calibration activity for audit readiness.
Red Flag #5: Drifting Baseline or Noisy Signals
An unstable or noisy baseline can be a sign of multiple system issues, many of which tie back to poor calibration or equipment performance.
Common Causes
Detector lamp degradation
Dirty flow cell
Improper mobile phase preparation
Air bubbles in the system due to pump leakage
Poor grounding or electronic interference
Why It Matters
A noisy or drifting baseline can obscure small peaks, alter integration results, and make it difficult to establish detection and quantitation limits, which is critical in impurity testing.
Solution
Schedule routine detector calibration, clean the flow cell, and degas solvents effectively. Maintain logs of lamp usage hours and replace UV lamps per manufacturer specs. Make sure the HPLC system is correctly grounded to minimize electrical noise.
The Cost of Ignoring Calibration
Operating an HPLC system without proper calibration can have serious consequences in biopharma labs:
Regulatory Non-Compliance: FDA, EMA, and other regulatory bodies expect documented evidence of regular calibration and maintenance. Failure to provide this can lead to Form 483 observations or Warning Letters.
Product Quality Risks: Misidentification or misquantification of impurities, APIs, or excipients can compromise product safety and efficacy.
Wasted Resources: Failed analyses lead to re-runs, wasting time, reagents, columns, and man-hours.
Data Integrity Violations: Inconsistent or unverified results may be flagged as integrity breaches, especially if audit trails and calibration logs are incomplete.
Best Practices to Verify Your HPLC Is Always Calibrated
To stay audit-ready and ensure consistent analytical quality, adopt a proactive approach to HPLC calibration:
Follow a Strict Calibration Schedule
Establish and follow a documented calibration plan based on manufacturer recommendations, regulatory guidance, and system usage levels. Include calibration of:
Flow rate
Detector linearity
Injection volume
Wavelength accuracy
Column oven temperature
Document Everything
Maintain clear, timestamped logs of calibration activities, SSTs, maintenance, and deviations. This documentation is critical for data integrity and regulatory compliance.
Use Certified Reference Materials (CRMs)
CRMs traceable to NIST or equivalent bodies validate that your calibration is scientifically valid and recognized by global regulatory agencies.
Train Lab Personnel
Make sure all analysts are properly trained in calibration procedures, method validation, and troubleshooting techniques. Calibration isn't just a checklist, it's a vital part of scientific rigor.
Leverage CMMS or LIMS Tools
Computerized Maintenance Management Systems (CMMS) or Laboratory Information Management Systems (LIMS) can automate calibration reminders, track instrument performance, and centralize documentation — improving both efficiency and compliance.
When to Recalibrate Your HPLC System
While regular intervals are important, you should also recalibrate any time the system undergoes:
Preventative maintenance or repairs
Major component replacements (e.g., detector lamp, pump seals)
Relocation or major environmental change
Method transfer from another lab or site
Unexplained deviation or out-of-spec results
Being proactive with recalibration reduces downtime and prevents compliance gaps.
Let's Wrap it Up!
In the high-stakes world of biopharmaceutical manufacturing and QC testing, HPLC calibration isn't a luxury, it's a necessity. From preserving data integrity to ensuring patient safety, every component of your HPLC system must function precisely and consistently. By watching for these five red flags (and taking immediate corrective action) your lab can uphold quality standards, meet regulatory expectations, and avoid costly errors.
About GL Technologies
GL Technologies, based in San Diego, is a specialized service provider catering to the highly regulated industries of biopharmaceuticals, pharmaceuticals, medical devices, and government sectors. The company focuses on delivering expert solutions in equipment calibration, validation, and compliance services, ensuring that clients meet stringent GMP (Good Manufacturing Practice) and FDA regulations. GL technologies is a trusted partner from commissioning new plants to decommissioning with compliance. GL can place dedicated motivated quality personnel on site anywhere. A program can be designed or revamped for the customers needs from design of CMMS to SOP development, specification development and performance of calibrations.
With a dedicated team of 29 technicians, GL Technologies offers precision calibration, preventative maintenance, and qualification services for laboratory and production equipment used in critical manufacturing and research processes. The company’s expertise is supporting its clients in maintaining regulatory compliance and operational efficiency.
As a full-service company specializing in equipment calibration, repair, and certification services for biopharmaceutical, pharmaceutical, and medical device industries. Our team has extensive experience working with sPRT calibrations along with CMMS software, HPLC OQ validation, and fume hood certifications. Companies of all sizes rely on our team to implement, maintain, and keep their research and manufacturing processes compliant with regulatory standards. Other specialties include building maintenance systems, and mass spectrometry calibrations. GL Tec specializes in IQ OQ PQ services for clients throughout San Diego, San Francisco, Los Angeles, Orange County, and Riverside!