Urine copper by colorimetry

Dear Editor,

The measurement of serum and urine copper is useful in assessing deficiency or toxicity. We recently validated the Randox (Parramatta, Australia) colorimetric assay for serum copper, ¹ and preliminary investigations also suggested that the assay might be suitable for the measurement of urine copper. We therefore undertook a more thorough evaluation of the assay. The Randox assay, which is based on the formation of a stable colour complex between copper and 4-(3,5-dibromo-2-pyridylazo)-N-ethyl-N-(3-sulfopropyl)aniline, was set up on the Binding Site, Optilite analyser (Edgbaston, UK). Sample, reagent, and chromogen volumes were 30, 125, and 30 µL, respectively. Incubation time was 300 s, and the reaction monitored at a wavelength of 575 nm. The sample was directly analysed against a calibration consisting of a reagent blank and a reconstituted lyophilized human urine calibration standard, ClinCal calibrator, copper concentration 2.3 µmol/L (Recipe, Munich, Germany). Recipe human urine ClinChek controls (copper concentration: 0.92 and 1.82 µmol/L) were used as internal quality controls (IQC). Intra- and interassay imprecision and accuracy were ascertained by the analysis of IQCs in quintuplicate on the same day and on different days (n = 5). Linearity and lower limit of quantitation (LLoQ) were assessed by non-serial dilutions of a patient urine sample (initial copper concentration: 15.0 µmol/L) with ultrapure water (>18.2 MΩ). Forty-eight urine quality assurance samples and 20 urine patient samples (24-h collections) were analysed. Results were compared to the median consensus, and those obtained by ICP-MS, respectively.

Intra-, and interassay accuracy was 70–84%, and 68–86%, respectively, and imprecision was 4–9%, and 3–7%, respectively. The assay was linear over the range 0.2–15.0 (R² = 0.99) µmol/L and the LLoQ was 0.2 µmol/L. For the quality assurance samples, there was an excellent correlation (rs = 0.99) but Passing–Bablok regression indicated a poor agreement (slope = 1.19, intercept = −0.16 μmol/L). The difference plot indicated that the median copper concentration (2.2 μmol/L) measured using the colorimetric assay was 0.2 μmol/L higher (P ≤ 0.05) compared to the median consensus value (2.0 µmol/L). For the patient samples, eight (40%) had copper concentrations below the LLoQ measured by colorimetry, which were all measurable by ICP-MS (median, range; 0.4, 0.2–1.6 μmol/L). For the remaining 12 samples, correlation was poor (rs = 0.88), as was agreement (slope = 0.39, intercept = −0.09 μmol/L). The median copper concentration (1.0, range 0.1–6.0 μmol/L) measured using the colorimetric assay was 1.1 μmol/L lower (P ≤ 0.05) compared to the median ICP-MS value (2.1, range 0.5–15.0 µmol/L).

For urine copper, the colorimetric assay had poor accuracy, and agreement with the median consensus values was also poor. In patient samples, comparison with ICP-MS was unacceptable, and in many samples copper could not be measured by colorimetry. The poor agreement in patient samples may be due to endogenous compounds interfering with analysis or that the copper concentrations were too low for accurate measurement. Nonetheless, the colorimetric assay for copper from Randox is not suitable for the measurement of urine copper in clinical samples.