Dear Stan and others interested,
> Wolfram,
> Could you point me toward the reference for the acetylene method. I have
> always been very curious about it.
> Thanks. Stan
>
Including yours, I have now received three enquiries as to the
acetylene method. So, I decided to Cc it to the discussion list, in
case there are others who might like to give it a go.
A word of caution though, a colleague and good friend of mine, who
is an old hand in all matters isotopic, tried it and got funny
numbers. We are not sure why this is. All I can hope is that it's not
associated with the batch / quality of the calcium carbide.
Here's the method we currently use and which works (so far):
A calcium carbide pellet (approx. 20 mg) [Aldrich Chemicals] is
placed into an Exetainer (screw top/septum glass tube, approx.
volume 11 mL). The Exetainer tube is evacuated and subsequently
filled with helium; 3-5 microliter of urine sample are carefully
pippetted onto the wall of the Exetainer tube (bottom half of tube
while the tube is held at an angle of >45 degree).
The tube is then closed, held upright and rotated around its
longitudinal axis to enable an even reaction between the calcium
carbide and the sample. We allow a reaction time of 30 minutes
(just to be on the safe side; the reaction is very fast; takes only
seconds, actually).
The acetylene gas thus generated is analysed by CF-IRMS.
Sample gas is carried by He (90 ml/min) through a water trap
(MgClO4) and purified on a 50 cm packed column (Carbon Sieve G;
1/4" O.D.) maintained at 125 C.
Isotope ratios are measured on a 20-20 IRMS (Europa Scientific),
monitoring mass ratio 27/26. On our instrument, HT is typically set
to 4180 V.
A standard calibration curve is run with each sample batch using a
serial dilution of 99.9% D2O.
There is no significant sample size dependence but the best
results are obtained from samples of 2 ul; S.D. is typically 0.0004
atom%, and coefficient of variation is typically 0.018%. For 4 ul
samples S.D. is 0.0006 atom% and cv is 0.029%.
As to the literature, Westerterp and Wagenmakers from
Maastricht published a paper in 1996. There is another 1996 paper
from a group in Cleveland where Beylot is one of the co-authors.
Here are the references:
(1) TI: DETERMINATION OF TOTAL-BODY WATER BY A
SIMPLE AND RAPID
MASS-
SPECTROMETRIC METHOD
AU: VANKREEL_BK, VANDERVEGT_F, MEERS_M,
WAGENMAKERS_T,
WESTERTERP_K,
COWARD_A
NA: UNIV LIMBURG HOSP,STABLE ISOTOPE RES
CTR,DEPT CLIN CHEM,POB
5800,6202 AZ MAASTRICHT,NETHERLANDS
MRC,DUNN NUTR CTR,CAMBRIDGE CB4 1XJ,ENGLAND
UNIV LIMBURG,DEPT HUMAN BIOL,6200 MD
MAASTRICHT,NETHERLANDS
JN: JOURNAL OF MASS SPECTROMETRY, 1996, Vol.31,
No.1, pp.108-111
IS: 1076-5174 AB: A rapid and inexpensive method was
developed
to determine
deuterium enrichment in body fluids. This is achieved by
converting water into acetylene. To vacutainer tubes a small
amount of calcium carbide is added. The tubes are
evacuated
and 25 mu l of sample are injected through the stopper. The
reaction takes place spontaneously at room temperature in a
few seconds. Enrichment at mass 27 compared with mass
26 can
be determined by continuous flow isotope ratio mass
spectrometry without any interference from the carrier gas
helium. A series of D2O samples diluted with increasing
amounts of H2O is prepared at the time of measurement of
the
biological samples and the measured ratios are used to
calculate the isotope dilution of the unknown. The relative
error of the method is 1.6% when a dose of 25 ml kg(-1) is
administered to the patient. The method was compared with
two different methods in nse in other laboratories, by a
published method. The means of the differences were -0.1
and
0.08 I, respectively, with standard deviations of 0.63 and
3.0.
KP: DOUBLY LABELED WATER, HYDROGEN
WA: D2O, TOTAL BODY WATER, MASS SPECTROMETER
(2) TI: ASSAY OF THE DEUTERIUM ENRICHMENT OF WATER
VIA ACETYLENE
AU: BRUNENGRABER_H, PREVIS_SF, HAZEY_JW,
DIRAISON_F, DAVID_F,
BEYLOT_M
NA: CASE WESTERN RESERVE
UNIV,CLEVELAND,OH,44106
INSERM,U197,F-69008 LYON,FRANCE
JN: FASEB JOURNAL, 1996, Vol.10, No.3, p.1228
IS: 0892-6638
DT: Meeting Abstract
(3) TI: ASSAY OF THE DEUTERIUM ENRICHMENT OF WATER
VIA ACETYLENE
AU: PREVIS_SF, HAZEY_JW, DIRAISON_F, BEYLOT_M,
DAVID_F,
BRUNENGRABER_H
NA: CASE WESTERN RESERVE UNIV,DEPT
NUTR,CLEVELAND,OH,44106
CASE WESTERN RESERVE UNIV,DEPT
NUTR,CLEVELAND,OH,44106
CASE WESTERN RESERVE UNIV HOSP,DEPT
SURG,INTEGRATED SURG
PROGRAM,CLEVELAND,OH,44106 INSERM U197,F-
69008 LYON,FRANCE
JN: JOURNAL OF MASS SPECTROMETRY, 1996, Vol.31,
No.6, pp.639-642
IS: 1076-5174 AB: A technique is presented for measuring the
H-2
enrichment of
water in biological samples when this enrichment is greater
than 0.2%. The sample is reacted with calcium carbide to
form acetylene gas, which is determined by gas
chromatography electron impact ionization mass
spectrometry.
Ion-molecule reactions, resulting in proton abstraction, are
minimized by lowering the electron ionization energy from
the usual 70 eV to 45 eV, This technique is much more rapid
and economical than the classical isotope ratio mass
spectrometric assay of the enrichment of hydrogen gas
derived from reduction of water.
KP: ISOTOPE RATIO MEASUREMENTS, SAMPLES, LIVER
WA: DEUTERIUM OXIDE, CALCIUM CARBIDE, ACETYLENE,
LIPOGENESIS,
GLUCONEOGENESIS, GAS CHROMATOGRAPHY MASS
SPECTROMETRY
Best regards,
Wolfram
*****************************************************
Dr. W. Meier-Augenstein, CChem MRSC
Senior Research Fellow
University of Dundee, Dept. of Anatomy & Physiology,
OMS, DUNDEE DD1 4HN, United Kingdom
Tel. (B): +44-(0)1382-34/5124 or /4574
Fax (B): +44-(0)1382-34/5514
e-mail (B): [log in to unmask]
URL: http://www.dundee.ac.uk/anatphys/wma/meieraug.htm
*****************************************************