Gas Chromatographic Analysis of Urinary Estrogens and its Identification with Gas Chromatography-Mass Spectrometry

Abstract

Analysis of estrogens in urine provids much important information on the endocrinological conditions of man related to his morbid conditions, the more precise method of which, however, is required today.
In the measurement of estrone, estradiol-17β and estriol several extracting methods, containing gas chromatographic analysis were reported and have been commonly used, However it was recently found by gas chromatography-mass spectrometry (Shimadzu LKB-9000) by the authors that they were not all adequate, producing too much contamination, so that we introduced a new methods in which each urinary estrogen can be accurately analysed with gas chromatography (Shimadzu GC-5APFE). With this method, estrone, estradiol-17β 78 and estriol were certified to be analysed in almost 100% in purities. In this paper our new extraction technique of estrogens from the human urine is reported.
Urinary estrogens were extracted as follows ; 300 ml of urine collected during 24 hrs. was used. After mixing with 210 g of ammonium sulfate and stirring for 40 min, urine was centrifuged at 8,000 × g for 40 min at 4°C. Precipitated urine was dissolved with 50 ml of water, adjusted with 0.2 M acetate buffer to pH 4.5, added to 100,000 Fishman Unit/800,000 Roy Unit of β-glucuronidase/aryl-sulfatase (Boehringer) and then hydrolysed for 21 hrs. at 37°C. Then, after adding 6 g of NaCl, estrogens were extracted twice with 100 ml of ether which were afterwards washed with 40 ml of sodium carbonate buffer solution (pH 10.5) and with 10 ml of saturated NaHCO₃ solution. After the evaporation, the extract was solved with 2 ml of methanol, and fractionated five times by an anion exchange column chromatography using AG1-X2 resin made by Bio-Rad. That is, after being washed with 20 ml of 0.5 M NaHCO₃ solution and 25.ml of methanol, the column was poured by 2 ml of estrogen containing methanol for five times. Then the column was washed with 15 ml and 25 ml of methanol to eliminate neutral steroids or impurities.
Estrogens were then extracted by 30 ml of 80% methanol and 20 μg of cholesterol was added as an internal standard, evaporated to dryness under 40°C. Trimethylsilylation was performed with N, O-Bis- (Trimethylsilyl) -acetamide to analyse estrogens with gas chromatography equipped with 1.5% OV-17 column. Gas chromatography-mass spectrometric analysis was also performed in parallel to identify estrogens. During these procedures several optical conditions were examined.
1) The optimum hour for hydrolysation of estrogens with β-glucuronidase/aryl-sulfatase was studied in 18, 20, 22, and 24 hours and it was found that hydrolysation for 21 hours exhibited the most effective recovery. The recoveries obtained in this condition are as follows ; estrone-3-sulfate 70.7±2.4%, estrone-3-glucuronide 74.5±3.8%, estradio1-17β3-glucuronide 74.8±3.5%, estriol-3-glucuronide 71.3±4.9% and estriol-16-glucuronide
2) The optimum unit of β-glucuronidase/aryl-sulfatase in hydrolysed for 21 hrs. was compared among 50,000/400,000,100,000/800,000,200,000/1,600,000,400,000 Fishman Unit/3,200,000 Roy Unit. More than 100,000 F.U./800,000 R.U. of enzyme hydrolysed more than 80.2±5.5% of conjugated estrogens.
3) The studies on recoveries of the concentration of various estrogen conjugates (0.5, 1.0, 2.5 and 5.0 μg) in the above-mentioned optimum conditions showed as follows;
estrone-3-sulfate 72.7±4.6%
estrone-3-glucuronide 83.1±6.3%
estradio1-17β-3-glucuronide 71.9±2.0%
estriol-3-glucuronide 80.0±4.4% and estriol-16-glucuronide 79.9±2.9%
The total recoveries of all of the estrogen conjugates exhibited 78.2±3.6% with this method.
Gaschromatography-mass spectrometric analysis revealed that the following estrogens were certified to be extracted with this method by gas chromatography;