Anal. Chem. 1987, 59, 690-693
690
Oxygen Isotope Ratio Measurements on Carbon Dioxide Generated by Reaction of Microliter Quantities of Biological Fluids with Guanidine Hydrochloride William W. Wong,* Lucinda S. Lee, and Peter D. Klein USDAIARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas 77030
Guanidine hydrochloride was used to convert water In biological flulds to carbon dloxlde for oxygen Isotope ratio measurements. Five 10-pL allquots each of five different saliva, urine, plasma, and human milk samples were allowed to react with 100 mg of guanidlne hydrochlorlde at 260 O C to produce ammonla and carbon dloxlde. Ammonla was removed with 100% phosphoric acid and carbon dioxide was cryogenically purified before Isotope ratio measurement. At natural abundances, the 6"O values of the biologlcal fluids were reproduclble to within 0.16%0 (standard deviation) and accurate to withln 0.11 f 0.73%0 ( a f SD) of the H,O-CO, equlllbratlon values. At a 250%0 enrichment level of "0, the 6l'O values of the biologlcal fluids were reproduclble to wHhln 0.95%0 and accurate to -1.27 f 2.25%0.
The guanidine hydrochloride method to convert water samples to carbon dioxide for oxygen isotope ratio measurements was reexamined recently by Dugan et al. ( 1 ) . In the followng reaction, the conversion of water to carbon dioxide is quantitative and the oxygen isotope composition of the carbon dioxide is a direct measure of the oxygen-18 content of the water samples:
NHZC:(NH)NHyHCl+ 2HzO
-+ 260 ' C
2NH3
COz + NH4C1 (1)
With the use of 100% phosphoric acid for the absorption of the ammonia produced by decomposition of ammonium carbamate and the strict control of reaction time, the P O values of the Vienna-Standard Mean Ocean Water (V-SMOW) and Standard Light Antarctic Precipitation (SLAP) were measured with a precision of 0.08 and 0.12%0(SD, n = 12), respectively. When compared with the Hz0-CO2equilibration values, those from the guanidine hydrochloride method were accurate to within -0.04 f 0.13%0(a f SD) for two laboratory water standards and 18 geothermal water samples. The small sample-size requirement of this method is attractive for nutrition studies of body composition or for estimates of energy expenditure in very low birth weight infants (2), small animals (3),or birds (4) in whom body fluid sampling is difficult or limited. Such samples also are likely to involve the measurement of enriched levels of oxygen-18. The effect of the biological fluid matrix and of enriched levels of l8O on the quality of the analytical results of the guanidine hydrochloride method must be determined, however, before the requisite sample size can be used to advantage. We describe a reaction assembly for the purification of the carbon dioxide generated from the guanidine hydrochloride reaction and our extension of the method to the analysis of microliter quantities of saliva, urine, plasma, and breast milk samples at natural abundances and enriched levels of oxy-
gen-18. The results were compared with those obtained by the H20-C02 equilibration technique and indicated satisfactory agreement.
EXPERIMENTAL SECTION Preparation of Aqueous Samples for Reaction. Standard 9-mm-0.d. Pyrex tubes were cut into 30-cm lengths and sealed at one end to form reaction tubes approximately 24 cm in length. These tubes were annealed a t 520 "C overnight, cooled to room temperature, and loaded with 100 mg of guanidine hydrochloride (Ultra-pure, Schwarz/Mann, Cambridge, MA). The reaction tube was attached to one side arm of a 3/8-in.Cajon Ultra-Torr tee (No. SS-8-UT-3, Cajon Co., Macedonia, OH); the other side arm was sealed off with a 9-mm-0.d. glass plug. The tee was evacuated through the center port to mbar. While under vacuum, the guanidine hydrochloride was melted with a heat gun. After the tube was cooled to room temperature, dry nitrogen was allowed to fill the tee and the reaction tube. A 2-mm-0.d. by 11-mm sample vial (Scientific Instrument Services, Inc., Ringoes, NJ) was loaded with a 1O-wL sample by using a Pencil-Slim micropipet (No. PS-10, Van Wilson, Lake Stevens, WA). The 9-mm-0.d. plug on the tee was removed, and under a flow of dry nitrogen, the sample vial was dropped into the reaction tube and the plug was replaced. The sample was frozen with liquid nitrogen and the reaction tube was evacuated to mbar and sealed, forming a tube approximately 20 cm long. The sealed tube was heated in a muffle furnace at 260 O C for 16 h to allow the sample to react with the guanidine hydrochloride (eq 1). At the end of the reaction period, the tube was removed from the furnace and allowed to cool to room temperature. Ammonium carbamate and ammonium chloride were formed as follows: 2NH3
+ COS
