B-012 Validation of a Urine Ammonium Assay on Roche Cobas c502 Chemistry Analyzer

Abstract

Abstract Background Urine ammonium (uNH4+) excretion is critical for the maintenance of acid-base balance and reduced uNH4+ excretion can cause retention of hydrogen ions resulting in metabolic acidosis in patients with chronic kidney disease (CKD). In addition, study found that declined urine ammonium excretion is an independent factor for predicting the worsening of kidney function. However, urine ammonium assay is not readily available on automated chemistry analyzers. We developed a protocol for the determination of urine ammonium levels using an ammonia assay for plasma samples on an automated chemistry analyzer. In this report, we present the urine ammonium protocol used in our laboratory and the validation of the urine ammonium assay. Methods All urine samples were diluted 100 times with deionized water and ammonium in the diluted samples was measured using the NH2L2 ammonia II assay on Roche cobas c502 chemistry analyzer. When a urine sample containing ammonium level less than 1.06 mmol/L, a 50-fold dilution was performed. Recovery was determined in six urine samples when 9 parts of the urine were mixed with one part of a standard 200 mmol/L ammonium solution (made with Ammonium Chloride from Thermo Scientific). Linearity was determined by serial dilutions of a sample containing 187 mmol/L ammonium. Limit of quantitation was determined using a 50-fold dilution protocol. The reference interval of 3–65 mmol/L was verified using 64 urine samples from volunteer donors and patient without CKD and metabolic acidosis. The stability was determined by testing two samples kept at room temperature for 0, 8, and 24 hour, respectively. The within-run precisions were determined by testing three urine samples 20 times in a run, and the between-run precisions were determined by testing three samples 2 times a day for 10 days, respectively. Results Recovery was 95.8% to 102.2% in six urine samples contained varying amount of ammonium ranged between 6.0 mmol/L to 22.2 mmol/L. Linearity was 0.5–99 mmol/L. Limit of quantification was 0.5 mmol/L. The reference interval of 3–65 mmol/L was verified. Urine ammonium was stable up to 24 h at room temperature. For within-run precision, the coefficient of variation (CV) was 1.51%, 0.49%, and 0.54% for samples containing 6.0 mmol/L, 27.7 mmol/L, and 75.7 mmol/L ammonium, respectively. For between-run precision, the CV was 2.54%, 5.98%, and 6.13% for samples containing 4.4 mmol/L, 27.9 mmol/L, and 55.1 mmol/L ammonium, respectively. Conclusion Urine ammonium levels can be determined using the NH2L2 ammonia II assay on Roche cobas c502 chemistry analyzer after manual dilution of urine samples 100 times with deionized water. The limit of quantitation is extended to 0.5 mmol/L when urine samples are diluted 50 times with deionized water.