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Evaluation of a high‐performance liquid chromatography method for urinary oxalate determination and investigation regarding the pediatric reference interval of spot urinary oxalate to creatinine ratio for screening of primary hyperoxaluria

Abstract Background Urinary oxalate can provide important clues for the screening and monitoring of children with primary hyperoxaluria (PH), which is a potentially life‐threatening condition. However, little effort has been devoted to improve the oxalate assay in recent years. We have proposed a reliable and cost‐effective high‐performance liquid chromatography (HPLC) method for urinary oxalate determination. Methods Urine specimens were centrifuged after one‐step derivatization, and the supernatants were subjected to HPLC analysis. Results The method was validated with consistent linearity from 0.0625 to 2.0 mmol/L with coefficients of variation ≤7.73%, good recovery, low carryover, satisfactory sample stability, and analytical specificity. The lower limit of quantification and the limit of detection were 0.03130 and 0.0156 mmol/L, respectively. Imprecision values were ≤2.92% and ≤16.6% for externally and internally produced controls, respectively. The pediatric reference interval of spot urinary oxalate to creatinine ratios was established together with its application in screening of PH in patients with renal diseases, revealing its successful deployment in our laboratory. Conclusions This reliable HPLC method could serve as a significant tool to determine urinary oxalate levels for screening and monitoring of children with PH in routine clinical laboratories.

from oxalate, nephrocalcinosis, and renal obstruction by stones. 4,5 The true prevalence of PH is unknown, and the estimated prevalence is less than three cases per million individuals. 4 Therefore, the rarity of this disease leads to a certain neglect in terms of screening and early diagnostic efforts. It is often diagnosed and concerned only after patients have experienced considerable pain over a prolonged period of time or even after they have reached end-stage renal disease (ESRD). For children, PH carries an increased risk of progression to ESRD as early as within the first few weeks of life and kidney or combined liver-kidney transplantation is always required to avoid death. 4,6 Considering the adverse and rapid progression of PH, testing of urinary oxalate, which is convenient in young children, is vital for biochemical screening and monitoring of PH.
A number of analytical methods such as enzymatic assays, 7,8 ion chromatography, 9 gas chromatography (GC), 10 high-performance liquid chromatography (HPLC), [11][12][13][14] and liquid chromatographytandem mass spectrometry (LC-MS/MS) 15,16 have been reported for the analysis of urinary oxalate. Among these, enzymatic assays are used by the majority of the laboratories, but a manual sample clean-up step with pH adjustment and activated charcoal columns is always required due to color inhibition caused by reducing substrates. 15 For GC, oxalate needs to be isolated initially by precipitation with calcium chloride and then derived for analysis. Although LC-MS/MS has recently gained increasing attention due to its high selectivity and sensitivity, it is not widely used when compared with HPLC due to its cost and complexity. 17,18 Consequently, interest in HPLC has been motivated, partially due to its low cost, easy operation, and ease of availability Nonetheless, HPLC methods described in the literature were developed 20 years ago and required large sample volumes, extensive sample preparation, specialized ion chromatography methods, or elution gradients, 14 accompanied by inadequate method evaluation. In addition to the aforementioned limitations, very few HPLC methods have been implemented in clinical laboratories and much less attention has been devoted to investigating the pediatric reference interval (RI), since various preanalytical and analytical factors related to different methodologies might lead to an extensive variation in the normal range of oxalate.
Therefore, our goal was to develop a robust and economical HPLC urinary oxalate assay along with an investigation regarding the RIs of spot urinary oxalate to creatinine ratio in children.
Ammonium acetate (LC-MS grade) was obtained from Fisher Scientific (Bremen, Germany). Methanol (HPLC grade) was from Merck (Darmstadt, Germany). Deionized water was prepared by a water purification system (ELga, High Wycombe, UK).

| Preparation of solutions
A stock solution of oxalic acid (40 mmol/L) was prepared in deionized water and stored at −80°C until analysis. In addition, a solution of OPD (0.46 M) in 2 M HCl was freshly prepared by dissolving 2.4 g of OPD in 40 mL of water, followed by mixing with 8 mL of HCl. A NaOH solution (10 M) was prepared by dissolving 40 g of NaOH in 100 mL of water.

| HPLC analysis
High-performance liquid chromatography coupled with an ultraviolet detector (Agilent 1200 Series LC System, Agilent Technologies, Santa Clara, CA, USA) was used for quantification. HPLC analysis was performed on a Phenomenex Luna C18 column (250 × 4.6 mm, particle size 5 μm) (Phenomenex, Torrance, CA, USA). Isocratic elution of 15% methanol in water containing 0.17 M ammonium acetate was used as the mobile phase for chromatographic separation at a flow rate of 1 mL/min. The column temperature was maintained at 25°C. The ultraviolet (UV) detector wavelength was set to 314 nm. After each sampling, the injection syringe was washed with water. All data were acquired using Agilent ChemStation software. From the peak area of oxalate recorded in the chromatogram and calibration curve plotted by peak area against calibration concentration, the concentration of oxalate in each sample was calculated by using the peak area over the slope of the calibration curve.

| Calibration, calibration verification, and quality controls
A stock oxalic acid solution (40 mmol/L) in a volumetric flask was prepared by dissolving 0.036 g of oxalic acid in 10 mL of water. were also obtained to construct matrix-matched calibrators, as described previously. A preliminary study was performed to determine whether there was any difference in the slopes of the oxalic acid calibration curves between calibrators prepared in water and those prepared in urine matrices. 19 A standard addition experiment was also performed by comparing the signals (peak areas) of the  (Table S1) showed no significant difference, and thus, water-based calibrators were used thereafter. Calibration verification was performed at three levels (0.10, 0.40, and 1.8 mmol/L) at least every six months using a commercially available standard with a different lot number. However, this was performed immediately in case of a change of reagent or column lot, unacceptable control values, or major maintenance performance (Clinical Laboratory Improvement Amendments-Calibration and Calibration Verification).
Quality controls (QCs) were prepared in two matrices (urine and water) and were labeled as internally produced controls (IQCs) and externally produced controls (EQCs), respectively. The IQCs were generated from pooled urine specimens and prepared at two levels within the expected biological range. The EQCs were prepared separately in water from a different lot number than those used as calibrators (0.250 and 1.000 mmol/L). The QCs were aliquoted and stored at −80°C for up to 1 month until use. QCs including both IQCs and EQCs were analyzed with a batch representing at least 5% of the total number of patient samples, which were always placed at the beginning, middle, and end of an analytical run.
All setups used for standard solution preparation including glassware, pipettes, and analytical balance were calibrated annually according to the College of American Pathologists checklist for our laboratory. All these descriptions have been documented in the "Standard Operating Procedure," and the corresponding operations have been routinely recorded in the "quality control management software" and the "daily record" in our laboratory for QC assurance.

| Sample preparation
Calibrators/QCs/urine specimens (1.0 mL) were mixed with 5 μL of concentrated HCl and 250 μL of OPD solution in tubes. The tubes were capped, vortexed, and heated to 120°C for 30 minutes. The solution changed from yellow to dark brown. After the solutions were cooled, 30 μL of 10 M NaOH was added. Finally, the solutions were centrifuged for 10 minutes at 18,000 rpm.

| Method evaluation
The European Medicines Agency guideline on bioanalytical method validation (European Medicines Agency 2011) was followed for evaluation of the present method emphasizing on linearity, the lower limit of quantification (LLOQ), the limit of detection (LOD), precision, accuracy, sample stability, carryover, and analytical specificity.

| Linearity
Five replicates were performed at each level of the calibrators in the range of 0.0625-2.0 mmol/L. An external calibration, in which the concentration of the analyte in a sample was calculated from its calibration curve plotted by the calibrators of this analyte, was used for oxalate determination.

| LLOQ and LOD
The LLOQ, defined as the lowest concentration with a signal-tonoise >10, was determined by assaying a series of low-concentration calibrators in five replicates and established based on the criteria that a coefficient of variation (CV) of <20% and an accuracy within 80%-120%. The LOD represents the concentration that gives a signal-to-noise >3.

| Precision
Two types of control materials made in water and in pooled urine (IQCs and EQCs) were used to evaluate precision. Five replicates of each level were assayed over five days to evaluate the intra-day and inter-day precision.

| Accuracy
Accuracy of the method was evaluated by a recovery study. Pooled

| Sample stability
The pooled urine specimen was aliquoted and stored at room tem-

| Carryover
A urine specimen spiked with 2.0 mmol/L of oxalic acid aqueous solution (95:5, v/v) was assigned as the high concentration sample, and water was assigned as the blank sample. The high concentration sample and the blank sample were injected in alternative for five times.
Carryover is not significant if the signal of any peak in the blank sample at the retention time of oxalate is below the signal of LOD.

| Analytical specificity
Analytical specificity was evaluated by assaying samples and QCs spiked with known concentrations of structurally similar compounds.
Citric acid, of which the property and the structure were similar to oxalic acid, was tested for analytical specificity. A citric acid aqueous solution (20 mmol/L) was spiked into a urine specimen, the low EQC and the high EQC at a ratio of 5:95. These samples together with an equivalent series of non-spiked samples were subjected to sample preparation for assaying interference.

| Urine collection and reference intervals
Spot urine specimens were collected without preservatives from pediatric subjects (n = 178, 114 males and 64 females, aged 2 days to 17 years, mean age: 4.5 years) to establish the RI of oxalate. The subjects were recruited and selected according to the following exclusion criteria: pediatric patients with renal diseases (kidney stones, hydronephrosis, nephritis, nephrotic syndrome) and digestive system diseases (idiopathic inflammatory bowel disease, ileectomy, jejunal/ileal bypass, celiac disease, chronic pancreatitis, chronic biliary diseases, and unexplained diarrhea with abnormal fat absorption). In addition, large doses (>2 g orally/24 hours) of vitamin C were avoided before specimen collection. All urine specimens were analyzed on the day of collection, and the concentrations of oxalate were measured using the newly proposed method. Urine creatinine (Cr) was determined using the Roche Cobas 8000 system (Roche, Germany).
Urinary oxalate was measured in 64 patients (38 males and 26 females aged 7 months to 17 years old with a mean age of 7.5 years) admitted to the pediatric nephrology unit of Tongji Hospital from June 2020 to August 2020. They were mainly characterized by renal disease, and screening for renal damage from PH was considered necessary. Finally, three cases were confirmed with PH.

| Statistical analysis
Normality of variable distribution was tested using Kolmogorov-Smirnov test or Shapiro-Wilk test. Continuous data were presented as mean (median). The RI was established using mean ± 1.96SD on the basis of normally distributed data; otherwise, the RI was presented in non-parametric 95th percentiles. Comparisons between two groups were made using t test or Mann-Whitney test, and comparisons among multiple-groups were performed by one-way ANOVA or Kruskal-Wallis test as appropriate. Pearson's test or Spearman's test was used to assess relationship among variables depending on normality of data. A linear model for an age-adjusted RI was generated by linear quantile regression analysis. All statistical tests were two-sided, and P values less than 0.05 were considered as statistically significant. The statistical analyses were performed using SPSS 13.0, Graphpad Prism 7.0, and Stata 12.0.

| Chromatography
During sample preparation, HCl was added. Oxalate primarily existed in the form of oxalic acid, which could react with o-phenylenediamine to yield the derivative 2,3-dihydroxquinoxaline ( Figure S1).
Considering the strong UV absorbance of this derivative, low concentrations of oxalate could be easily detected. Quantitative chromatographic profiles of a standard solution, a spiked urine sample, urine sample from a healthy child, and urine sample from a child with PH are presented in panels A, B, C, and D of Figure 1. The retention time was approximately 13 minutes, and all samples achieved excellent separation without interference peaks. After the chromatographic column was injected 800 times, the theoretical plate number of this column was 7638, which was consistent with its initial performance. The target analytes in urine specimens were identified by comparing their retention times with those of the calibrators and controls.

| Linearity
In this experiment, water-matched and matrix-matched calibrators were prepared. The slopes of the calibration curves of the two types of calibrators were not significantly different, with percentage differences in the range from -5.57% to 1.23% (Table S1). The percentage differences in peak areas in the standard addition experiment were ≤2.66% (Table S2), demonstrating that the signals of the analytes in water and in urine showed no obvious differences. Hence, water-based calibrators were used for validation.
The inter-assay (n = 5) variability of the calibration data was monitored. CV values were ≤7.73% (Table 1)

| LLOQ and LOD
Analysis of a series of low-concentration calibrators (0.00195, 0.00390, 0.00780, 0.01560, and 0.03130 mmol/L) resulted in a CV of <20% and an accuracy of 80-120% at a concentration of 0.03130 mmol/L. Therefore, the concentration of 0.03130 mmol/L, which provided a 10-fold higher signal than that of noise, was accepted as the LLOQ. The LOD, defined as the concentration with a signal threefold higher than that of noise, was 0.0156 mmol/L.

| Accuracy
Recovery was performed in five replicates for each sample by spiking known amounts of oxalic acid into urine specimens. The recoveries

| Sample stability
The fresh pooled urine was stored at room temperature for at least 7 days with a CV of 6.24%. At 4°C and −80°C, the storage time of the pooled urine extended to at least 14 days with CVs of 13.1% and 7.20%, respectively, revealing that the urine samples could be stored at 4°C or lower for a longer storage duration.

| Carryover
The high concentration sample and the blank sample were injected in alternative for five times. No peaks were found in blank samples at the retention time of oxalate, demonstrating that no carryover was presented.

| Analytical specificity
Citrate exerts an inhibitory effect on the formation of calcium oxalate stones and has a similar structure to oxalate. 20 Citric acid was tested for analytical specificity. Percent bias, calculated as the ratio of the analyte concentrations in the spiked vs. baseline samples, was employed to evaluate interference. 21 After the addition of citric acid to the sample, the low control, high control, and average biases of oxalate were 4.96%, 2.38%, and 1.80%, respectively. Furthermore, no interference peak was observed at the retention time of oxalate.

| Reference intervals
The pediatric RI of spot urinary oxalate to creatinine ratio was inves- A wide variety of analytical procedures and methods used for urinary oxalate determination might lead to substantial batch-tobatch and lab-to-lab variations. Hence, the RI was investigated based on the determination of urinary oxalate using the present HPLC method. Since diet composition might influence urinary oxalate excretion, 22 infants up to 6 months of age who exclusively consume milk are more likely to be included in an age group alone. Furthermore, according to the scatter plot of oxalate/Cr vs. age presented in Figure 2 and the median splits presented in Table 4, pediatric subjects were divided into four age subgroups (<0.5, 0.5-2, 2-8, and 8-17 years of age,

| Clinical testing
The present method offered an excellent opportunity for use in our laboratory. To date, 64 patients with suspected PH have been subjected to clinical testing for oxalate in spot urine specimens.
Among these, three cases were confirmed to have PH. These pa-  In addition, a comparison study between the calibrators prepared in water and in urine, which focused on the slope of the calibration curve and signal intensity, showed no significant differences, alleviating the difficulty in preparing matrix-matching calibrators from oxalate-free urine. Moreover, this method has already been shown to be robust in routine use, with no batch failures to date.

| DISCUSS ION
A variety of sample preparation procedures and analytical techniques have been employed in urinary oxalate assays, resulting in uncertain effects on testing results due to the lack of global standardization in these laboratory developed tests. Therefore, establishment of an RI for urinary oxalate in children is of utmost importance and offers excellent promise for its use in clinical laboratories. The scatter plot of oxalate/Cr vs. age is presented in Figure 2.
The study population was initially divided into seven specific groups.
Subsequently, based on median splits presented in Table 4  Moreover, external quality assessment is urgently needed to assess the performance of this method in a clinical laboratory.

| CON CLUS ION
We successfully developed and validated an HPLC method for the determination of urinary oxalate levels. Due to the reliability, simplicity, and cost-effectiveness of this method, it would be suitable as a screening and monitoring tool for children with PH.

ACK N OWLED G M ENT
We gratefully acknowledge the financial support from the National Natural Science Foundation of China NSFC-81401718.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data can be available if needed.

R E FE R E N C E S
The data are abnormal distribution, and the reference intervals are expressed in non-parametric 95th percentiles.