Quantification of Six Porphyrin Biomarkers in Urine Using LC-2050C with Fluorescence Detection

Significance of the Topic

Porphyria disorders arise from defects in heme synthesis leading to accumulation of porphyrin intermediates. Reliable quantification of urinary porphyrins is critical for early diagnosis, monitoring of disease progression, and differentiation among porphyria subtypes. High‐performance liquid chromatography with fluorescence detection provides the selectivity and sensitivity needed for clinical and research laboratories.

Objectives and Study Overview

This study presents a straightforward, robust HPLC method for simultaneous measurement of six urinary porphyrins: uroporphyrin I, heptacarboxyporphyrin I, hexacarboxyporphyrin I, pentacarboxyporphyrin I, coproporphyrin I, and coproporphyrin III. The goal was to develop a protocol requiring no sample pre-treatment while maintaining high accuracy and precision.

Methodology and Instrumentation

The protocol employs a commercial lyophilized porphyrin standard kit containing calibrator mixtures and two control levels. Standards were reconstituted in water and serially diluted in mobile phase under dark conditions. Urine controls were injected directly. Analytical conditions included:

• Column temperature 25 °C, autosampler at 4 °C
• Shim-pack Scepter C18-120 (250 × 4.6 mm, 5 µm)
• Mobile phase A: 10 mM ammonium acetate (pH 5.6), B: acetonitrile
• Gradient elution from 0 % to 45 % B over 17 min, return to 10 % B by 17.1 min, total runtime 25 min
• Flow rate 1.0 mL/min, injection volume 20 µL
• Fluorescence detection at excitation 395 nm and emission 630 nm

Main Results and Discussion

Calibration curves for all six porphyrins demonstrated excellent linearity with correlation coefficients exceeding 0.999. Accuracy for calibration levels ranged from 94 % to 117 %, with precision (%RSD) for retention time below 0.07 % and concentration below 0.84 %. Analysis of two urine control samples (n = 6) yielded measured concentrations within specified ranges and accuracy between 94 % and 108 %. These results confirm the method's reliability for routine porphyrin measurement.

Benefits and Practical Applications

• No sample pre-treatment simplifies workflow and reduces analysis time.
• Robust performance on widely available HPLC instruments.
• High sensitivity and specificity via fluorescence detection.
• Cost-effective approach suitable for clinical laboratories.
• Supports differential diagnosis of porphyria subtypes in patient samples.

Future Trends and Potential Applications

Emerging directions include integration of automated sample handling, coupling with mass spectrometry for confirmation, miniaturized flow systems for point-of-care testing, and real-time monitoring of porphyrin excretion in clinical studies. In addition, expanding the method to include additional porphyrin isomers or related metabolites could further enhance diagnostic capabilities.

Conclusion

A simple, low-cost HPLC-fluorescence approach has been validated for quantifying six key porphyrin biomarkers in urine without sample preparation. The method exhibits excellent linearity, accuracy, and precision, making it attractive for clinical and industrial laboratories aiming to streamline porphyria testing.

References

• Tupe D, Trivedi B, Sutar P, Rasam P, Kelkar J. Quantification of Six Porphyrin Biomarkers in Urine Using LC-2050C with Fluorescence Detection. Shimadzu Analytical (India) Pvt. Ltd.; 2023.