Colorimetric method for determination of urinary methylmalonic acid

Area of Science:

• Analytical Chemistry
• Clinical Biochemistry

Background:

• Methylmalonic acid (MMA) is a biomarker for cobalamin (vitamin B12) deficiency.
• Accurate and sensitive quantification of MMA is crucial for diagnosing metabolic disorders.
• Existing colorimetric methods for MMA determination require optimization for improved performance.

Purpose of the Study:

• To re-examine and improve the colorimetric method for methylmalonic acid determination.
• To enhance the stability, sensitivity, and reliability of the assay.
• To adapt the method for routine use with small urine volumes.

Main Methods:

• Re-examination of the colorimetric method involving coupling with diazotised p-nitroaniline.
• Extraction of the diazotised product using amyl alcohol and tetra-methyl ammonium hydroxide.
• Preliminary solvent extraction and ion-exchange chromatography for sample preparation.

Main Results:

• Optimized extraction significantly increased the stability and sensitivity of the assay.
• Background absorbance was effectively reduced.
• The method demonstrated high reliability with approximately 95% recovery.
• A low coefficient of variation of about 7% was achieved.

Conclusions:

• The enhanced colorimetric method provides a reliable and sensitive means for determining methylmalonic acid in urine.
• The improved assay is suitable for clinical diagnostics, even with minimal sample quantities.
• Further validation could establish this method as a standard for MMA quantification.