Background High-Performance Liquid Chromatography (HPLC) coupled with fluorescence detection has been the gold standard for the differential diagnosis of porphyria for nearly 40 years. Analysing porphyrins in urine is a robust way to identify most forms of porphyrias. In the age of looming In Vitro Diagnostics Regulation (IVDR), the development of in house HPLC methods remains for the most adept clinical chemistry laboratories. Fortunately, companies like Chromsystems are making IVDR kits for the analysis of porphyrins in urine by HPLC. The North Estonia Medical Centre laboratory has recently incorporated the aforementioned kit and tried to verify the manufacturers claims.

Materials and Methods Verification of the Chromsystems Porphyrins in Urine IVDR kit was performed on a Shimadzu LC-20A Prominence HPLC system equipped with an RF-20A fluorescence detector. All required and optional components of the kit were used including the analytical column, precolumn and the prefilter. Samples were separated into uroporphyrin, heptacarboxyporphyrin, hexacarboxyporphyrin, coproporphyrin I, coproporphyrin III and quantified using a calibrator and an internal standard. Precision (repeatability and within-laboratory precision) was assessed using internal quality controls (IQC) (n=14 for each analyte) and evaluated with Analysis of Variance (ANOVA) statistics against the manufacturers reported Coefficient of Variation (CV) and the target ranges of the IQC materials. Accuracy was established by participating in external quality assessment (EQA) scheme by Instand.

Results The ANOVA statistics showed that we were not able to achieve the same precision results with all parameters as the manufacturer of the kit. Chromsystems had results with a CV of 0.5–1.6% for repeatability and 1.5–2.4% for within-laboratory precision while our numbers were within a range of 0.5–13% and 7.8–17% respectively. However, the results were well within the target ranges of the control materials. Participation in the INSTAND EQA show that we were able to get similar results as others within our group as the average difference was 2.4%.

Conclusions Even though we did not achieve the same results as the manufacturer of the kit, our results were far from unacceptable. Discrepancy was likely due to using lyophilized urine control materials instead of freezing aliquots of a single sample. EQA results and patient cases prove that this kit is producing reliable results. Chromsystems has taken a good step forward to provide a simple to implement method to separate and identify relevant porphyrins in urine. Unfortunately, kits for the analysis of different biological matrices for porphyrins are hard to come by, using only urine, as a diagnostic tool does not cover the entirety of porphyria diagnostics. To rule out all forms of porphyrias, laboratories must either forward a sample to a different laboratory or delve into the world of developing an in house method for either plasma, blood or stool.