title: Determination of urine oxalate level in rats with renal calcium oxalate calculus by high-performance liquid chromatography.
abstract: OBJECTIVE
  To establish a method of high-performance liquid chromatography (HPLC) for determining the urine oxalate levle in rats with renal calcium oxalate calculus.
  
  
  METHODS
  Totally 24 SPF Wistar healthy male rats were randomly divided into control group(n=12)and ethylene glycol (EG) group (n=12). Rats in EG group were administered intragastrically with 2% ammonium chloride (AC)2 ml/rat per day+1% ethylene glycol (EG), along with free access to drinking water.The control group was fed with deionized water, along with the intragastric administration of normal saline (1 ml per day). Twenty-eight days after modelling, the 24-hour urine samples were collected, and the urine oxalic acid levels were determined using HPLC and the results were compared with those of catalytic spectrophotometry using oxidation of methyl. During the HPLC, the samples were separated on Aglient 5TC-C18 (250×4.6 mm,5 Μm), eluted with mixture of methanol (0.1 mol/L) and ammonium acetate (15:85) at 1.2 ml/min, and detected at 314 nm, with the column temperature being 20 ℃.
  
  
  RESULTS
  The standard curves of high and low concentrations of oxalic acid were y=5909.1x+378730, R² =0.9984 and y=7810.5x-16635, R² =0.9967,respectively. The lowest detectable concentration in this method was 5 Μg/ml. The linear high concentration range of oxalate stood at 62.50-2000.00 Μg/ml, and the linear low concentration range of oxalate stood at 6.25-100.00 Μg/ml. Its average recovery was 95.1%, and its within-day and day-to-day precisions were 3.4%-10.8% and 3.8%-9.4%. Both HPLC and catalytic spectrophotometry showed significantly higher urinary oxalic acid concentration and 24 h urine oxalate level in EG group compared with the control group [urinary oxalic acid concentration: (736.35 ± 254.52) Μg/ml vs.(51.56 ± 36.34) Μg/ml,(687.35 ± 234.53) Μg/ml vs.(50.24 ± 42.34) Μg/ml;24 h urine oxalate level: (11.23 ± 4.12)mg vs.(0.87 ± 0.45)mg,(9.89 ± 3.55)mg vs. (0.77 ± 0.65)mg; all P<0.01]. No statistically significant difference was observed in the results of urinary oxalate concentration and 24 h urine oxalate level between HPLC and potassium chromate oxidation of methyl red spectrophotometry (all P>0.05).
  
  
  CONCLUSIONS
  HPLC is a simple, rapid, and precise method in detecting urine oxalate level in rats with renal calcium oxalate calculus, with high recovery rate.
authors: Clinical Medical College of Traditional Chinese Medicine, Hubei University of Chinese Medicine, Wuhan 430061, China.
authors: Qiu-shi
authors: QS
authors: Cao
authors: Department of Nephrology, Hospital of Traditional Chinese Medicine in Huibei, Wuhan 430061, China.
authors: Yuan-ming
authors: YM
authors: Ba
authors: Clinical Medical College of Traditional Chinese Medicine, Hubei University of Chinese Medicine, Wuhan 430061, China; Department of Geriatrics, Wuhan General Hospital of PLA Guangzhou Military Command, Wuhan 430070, China.
authors: Jun-hua
authors: JH
authors: Luo
authors: School Hospital, Hubei University of Chinese Medicine, Wuhan 430061, China.
authors: Qi
authors: Q
authors: Dai
mesh_terms: D000085:Acetates; D000818:Animals; D001391:Azo Compounds; D002129:Calcium Oxalate; D002137:Calculi; D002840:Chromates; D002851:Chromatography, High Pressure Liquid; D007668:Kidney; D008297:Male; D010070:Oxalates; D017680:Potassium Compounds; D051381:Rats; D017208:Rats, Wistar; D013053:Spectrophotometry; D014867:Water
chemical_list: D000085:Acetates; D001391:Azo Compounds; D002840:Chromates; D010070:Oxalates; D017680:Potassium Compounds; D014867:Water; D002129:Calcium Oxalate; C027373:potassium chromate(VI); C008492:methyl red; C018824:ammonium acetate