Spot-on UV/Vis accuracy with Lunatic & Stunner

Importance of the Topic

Verifying UV/Vis spectrometer performance is essential for reliable quantification of proteins and nucleic acids in biologics research and quality control. Rapid, accurate instrument qualification saves time and resources by ensuring measurements meet regulatory and scientific standards before large‐scale studies begin.

Study Objectives and Overview

This work assesses the accuracy, precision, and linearity of Unchained Labs’ Lunatic and Stunner microvolume UV/Vis instruments. Performance was evaluated using: certified reference materials from NIST (RM 8671 monoclonal antibody, SRM 2082 tryptophan and uracil standards), the Fundamentals L-tryptophan kit, and gravimetrically prepared protein dilution series. Goals included full‐range absorbance verification at 260 and 280 nm and demonstration of inter- and intra-instrument reproducibility.

Instrumentation Used

The main instruments were:

• Lunatic microvolume UV/Vis spectrometer: fixed-pathlength microcuvettes, 2 µL sample, up to 96 samples per run.
• Stunner instrument: combines UV/Vis spectral analysis with dynamic light scattering in micro-volume plates.
• Fundamentals Check kit: five certified aqueous L-tryptophan concentrations for absorbance 230–310 nm qualification.
• NIST reference materials: RM 8671 human IgG1K for concentration accuracy; SRM 2082 tryptophan and uracil for pathlength validation.

Methodology

NIST Evaluation
• Five Lunatic units; octuplicate measurements of 10 mg/mL NISTmAb at 280 nm.
• SRM 2082 tryptophan (1.4 mM) and uracil (1.0 mM) standards at 260/280 nm to calculate pathlength vs. nominal 10 mm.

Fundamentals Kit Assessment
• L-tryptophan standards (1–8 mg/mL) in octuplicate using Lunatic & Stunner v7.0 software.
• Linear regression of measured vs. target concentrations to evaluate accuracy, precision, linearity.

Protein Dilution Series
• Human serum albumin (0.323–365.5 mg/mL) and polyclonal IgG (0.1–200 mg/mL) prepared gravimetrically.
• Measurements on five Lunatic instruments with quick-load and standard protocols.

Main Results and Discussion

NIST Standards
• NISTmAb concentrations were within ±1.25% bias of the 10 mg/mL reference.
• SRM 2082 pathlengths deviated by less than 2% from nominal.

Fundamentals Kit
• Linear regression slope ≈0.9945, R² = 0.9999 across 1–8 mg/mL tryptophan.
• Mean recoveries between 99.1% and 100.6%, RSDs below 1%.

Protein Series
• Inter-instrument %CVs for HSA samples below 1.5% across 230–310 nm.
• IgG in PBS and acetate buffers showed linear concentration response from 0.1 to 200 mg/mL with R² ≈ 0.9999.

Benefits and Practical Applications

• Fast, low-volume (<2 µL) qualification simplifies routine instrument checks.
• High throughput (up to 96 samples) supports screening workflows.
• Certified standards and automated software reporting meet QA/QC and regulatory requirements.

Future Trends and Applications

• Expanded use of certified microvolume kits for additional wavelengths and analytes.
• Integration with Laboratory Information Management Systems for audit-ready records.
• Adoption in regulated biologics development and manufacturing environments.

Conclusion

Lunatic and Stunner UV/Vis spectrometers deliver robust accuracy, precision, and linearity across a wide concentration range. Validation with NIST reference materials and the Fundamentals kit confirms performance within tight bias and variability limits, streamlining instrument qualification in research and quality control labs.

References

• National Institute of Standards and Technology. RM 8671 NISTmAb Humanized IgG1K Monoclonal Antibody (2020).
• National Institute of Standards and Technology. SRM 2082 Pathlength Absorbance Standards for Microliter Volume Spectrophotometers (2020).
• United States Pharmacopeia. Chapter <857> Ultraviolet-Visible Spectroscopy, USP 42.