QUANTITATIVE MEASUREMENT OF DRIED BLOOD SPOT GUANIDINOACETATE AND CREATINE FOR CLINICAL RESEARCH

Importance of the Topic

Newborn screening for guanidinoacetate methyltransferase (GAMT) deficiency addresses a critical inborn error of metabolism leading to cerebral creatine depletion. Early detection of elevated guanidinoacetate (GUAC) and reduced creatine (CRE) concentrations in dried blood spots (DBS) can prevent severe neurological impairments through prompt intervention. Incorporation of GUAC and CRE into routine screening improves diagnostic coverage for creatine synthesis disorders.

Objectives and Study Overview

This study aimed to establish and validate a non-derivatized flow-injection analysis tandem mass spectrometry (FIA-MS/MS) method for quantifying GUAC and CRE in DBS. Specific goals included assessment of calibration linearity, precision, recovery, and accuracy against external CDC reference materials, following CLSI and CDC guidelines.

Methodology

Stable isotope labeled GUAC-d2 and CRE-15N3 internal standards were spiked into extracts from 3.2 mm DBS punches prepared on Whatman 903 filter paper. In-house calibrators (levels L1–L9) were generated by mixing blood adjusted to endogenous concentrations with enriched samples up to the upper linearity limit. Extraction proceeded at room temperature in a 96-well plate, injecting 10 µL directly into the MS/MS system without derivatization. Transitions monitored in positive ESI MRM mode were 118.1>76 for GUAC and 132.1>90 for CRE, with corresponding labeled standards.

Used Instrumentation

• Waters MassTrak ACQUITY UPLC I-Class PLUS system
• FL Sample Manager
• Xevo TQD Mass Spectrometer
• Analyse-IT software for data processing

Main Results and Discussion

Linearity experiments across nine levels demonstrated r² ≥ 0.99 for both GUAC and CRE, with allowable deviation under 20%. Precision evaluated by four replicates per level showed imprecision below 20%. Recovery averaged 108% for GUAC and 91% for CRE. Comparison with external CDC linearity samples yielded accuracy of 96% for GUAC and 105% for CRE, confirming strong correlation to reported values.

Benefits and Practical Applications

• Robust quantitation of GUAC and CRE supports multi-tiered newborn screening strategies.
• Non-derivatized workflow reduces preparation time and chemical use.
• High throughput FIA-MS/MS enables rapid analysis of routine DBS samples.

Future Trends and Potential Uses

Anticipated developments include expansion of non-derivatized assays to additional metabolic markers, integration into automated newborn screening platforms, and application of high-resolution MS for enhanced specificity. Emerging bioinformatic tools may further streamline data interpretation and cross-laboratory harmonization.

Conclusion

The described non-derivatized FIA-MS/MS method on the Waters MassTrak Xevo TQD system delivers reliable, linear, and accurate measurement of GUAC and CRE in DBS. Its performance aligns with CDC standards, supporting its adoption in clinical research and potential translation into routine newborn screening workflows.

References

• Baby’s First Test, 2022. Conditions: Guanidinoacetate Methyltransferase Deficiency.
• Kilgore et al., 2023. Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids. Anal Chem. 95(6):3187–3194.