High Throughput Testing Method with Newborn’s DBS for Hemoglobinopathies by LCMS-8045 with ZenTech Targeted MS/MS Hemo Device

Significance of the Topic

This work addresses the rapid and precise identification of hemoglobin variants in newborn dried blood spots (DBS). Early detection of hemoglobinopathies such as sickle cell disease and thalassemias is critical for timely clinical intervention and improved patient outcomes. Traditional methods can be time-consuming, pH-sensitive, and prone to false results. A high-throughput mass spectrometry approach offers robustness, specificity and scalability to meet the demands of large-scale newborn screening programmes.

Objectives and Study Overview

The primary aim was to implement and validate a targeted LC-MS/MS workflow on the Shimadzu LCMS-8045 coupled with the ZenTech Targeted MS/MS Hemo device. The study focused on the detection and quantitation of α- and β-globin peptides corresponding to normal (HbA) and clinically relevant variants (HbS, HbC, HbE, HbOArab, HbDPunjab). Key goals included achieving sub-one-minute analysis time per sample, demonstrating reproducibility across routine DBS specimens, and correlating results with known phenotypes.

Methodology and Instrumentation

Sample preparation involved extraction and denaturation of hemoglobin from DBS punches, followed by tryptic digestion. Internal controls supplied in the ZenTech kit ensured consistency of extraction, denaturation and digestion steps, and monitored instrument performance.

• Liquid Chromatograph: Nexera X3 UHPLC system, isocratic elution (50% 0.1% formic acid in water / 50% 0.1% formic acid in acetonitrile), 0.4–0.6 mL/min flow, 5 µL injection.
• Mass Spectrometer: Shimadzu LCMS-8045 triple quadrupole with positive ESI, ion interface 3.5 kV, DL and heat block at 200 °C, interface at 400 °C, CID gas 270 kPa.
• Acquisition Mode: Multiple Reaction Monitoring (MRM) targeting unique α- and β-globin peptides and variant-specific transitions.

Main Results and Discussion

Analysis of over 500 routine DBS samples yielded clear chromatographic peaks with retention times under one minute. The method correctly identified normal samples and carriers of HbS, HbE and HbDPunjab, matching expected phenotypes (Table of 508 normals, 5 HbS carriers, 1 HbE carrier, 1 HbDPunjab carrier). Peak shapes were sharp and reproducible, and no false positives or negatives were observed. The targeted MRM transitions enabled precise discrimination of variants that are challenging to resolve by conventional electrophoresis or HPLC.

Benefits and Practical Applications

• High Throughput: <1 min analysis time accelerates large-scale screening.
• Robustness: Stable performance across routine DBS matrices.
• Sensitivity and Specificity: Accurate detection of low-abundance variant peptides.
• Integration: End-to-end workflow from sample prep to data output suitable for clinical laboratories.

Future Trends and Opportunities

Advancements may include automated DBS handling, multiplexed assays for additional genetic markers, and integration with digital laboratory information systems. Emerging high-resolution MS technologies and AI-driven data analysis could further enhance throughput and variant discovery. Expanding this targeted approach to other protein disorders holds promise for newborn and population screening.

Conclusion

The Shimadzu LCMS-8045 combined with the ZenTech Targeted MS/MS Hemo device provides a fast, sensitive and reliable solution for newborn hemoglobinopathy screening. The method’s performance in routine DBS samples demonstrates its suitability for high-volume laboratories, offering clear advantages over conventional techniques.

References

1. ADAM Medical Encyclopedia. Thalassemia. A.D.A.M., Inc.; updated 2014 Feb 24.
2. Burtis CA, Ashwood ER, Bruns DE, editors. Tietz Fundamentals of Clinical Chemistry. 6th ed. Elsevier; 2008.
3. National Library of Medicine. HBB gene. 2009.
4. AACC. Hemoglobinopathies. Higgins T. Dec 29, 2014.