Anal. Chem. 1985, 57,469-471
469
Stable Isotope Ratio Mass Spectrometry for Iron Bioavailability Studies John Eagles, Susan J. Fairweather-Tait, and Ron Self* Food Research Institute, Colney Lane, Norwich, U.K.
Purpose-built inorganic isotope ratio mass spectrometers are not yet readily available in biological laboratories, whereas medium resolution organic mass spectrometers equipped with fast atom bombardment sources are becoming widespread. A method is described for the measurement of the 58Fe/58Fe ratio, with an “organic” mass spectrometer, providing sufficient accuracy and precision for iron incorporation studies on human fecal samples. Only a relatively simple solvent extraction of the ashed material is required to provide a sample free from isobaric interference from nickel. The amount of iron absorbed from 10 mg of 58Fe-iabeiedferrous sulfate was Calculated as the difference between fecal ”Fe (unabsorbed) and the oral dose of 58Fe.
The absorption of iron (Fe) from food depends upon the physiological status of the individual, the chemical form of the Fe in the food, and the presence of other food components in the intestine which may either potentiate or inhibit the absorption of non-heme Fe ( I , 2). For example, there is some concern over the increasing substitution of vegetable protein for meat, since the former contains Fe which is less readily absorbed (3). The most accurate and sensitive method of studying mineral absorption and metabolism uses radioisotopes as labels and employs whole body counting techniques ( 4 ) . However, the use of 59Fe, a high energy y emitter, is considered to be unethical for a t least two nutritionally important groups of people, pregnant women and infants, and, therefore, a stable isotope alternative has been tested and found to be acceptable ( 5 ) . Non-heme iron absorption from a food can be determined by extrinsically labeling the endogenous iron with an enriched source of %Feand measuring the amount excreted in the feces. The amount absorbed can then be deduced subtractively. Neutron activation analysis (NAA) was used to measure total j8Fe, and the level of naturally occurring 58Fewas calculated from the total Fe determined by atomic absorption spectrophotometry (AAS). The success of this approach depends heavily on subject cooperation in making accurate total fecal collections. The preferred method of making direct measurements on blood samples is more difficult when using NAA which is less sensitive than the conventional method of y counting, and NAA suffers two further disadvantages which have restricted our progress, namely, access to a suitable reactor and the slow turn around (in excess of 4 weeks) of samples. A more sensitive, interactive, and accessible method is sought to facilitate a full program of work on the incorporation of Fe in blood, saliva, human milk, and other tissues as well as in feces. The measurement of stable isotope ratios of inorganic elements by mass spectrometry is only new in its routine application to biological samples. In this rapidly growing area, methods have been described for Fe which employ thermal ionization (6) and secondary ion mass spectrometry (7) for direct measurements and electron ionization ( 4 9 ) after initial conversion to a volatile derivative. Although thermal ioni-
Table I. Natural Abundances (NA) of the Stable Isotopes of Irono isotope
70 NA
at mass
% RI
54 56
5.82 91.66 2.19 0.33
53.9396 55.9349 56.9354 57.9333
6.35 100 2.39 0.36
57
58
a58Fe/56Fe isotope ratio = 0.0036. zation mass spectrometers are undoubtedly the most accurate for isotope ratio measurement, they are not readily available yet in the biological and nutritional areas, and, therefore, methods utilizing conventional instruments are being developed. Our early experience (10) of fast atom bombardment (FAB) mass spectrometry (11)showed that beams of inorganic ions of reasonable stability could be produced easily via the glycerol matrix, but their low concentration, at a resolution high enough to remove all organic and inorganic interferences, was a limiting factor. When Smith (12) demonstrated that for monoatomic analytes, such as calcium, the dispersing medium was not required, we decided to investigate his method for the measurement of the degree of incorporation of iron in human subjects, by feeding iron enriched in isotope 58 (Table
I). We report the use of selected ion monitoring medium resolution FAB, using a conventional organic mass spectrometer, for the direct and rapid measurement of the 58Fe/56Fe isotope ratio (Table I) for ashed fecal samples, requiring only a simple solvent extraction in preparation for accurate analysis.
EXPERIMENTAL SECTION Fasting human subjects were given a solution of ferrous sulfate containing 10 mg of Fe, extrinsically labeled with 1.31 mg of 58Fe (enrichment factor 72.18% AERE, Harwell). Feces were collected until all the unabsorbed jsFe had been excreted (transit time was measured by using carmine markers given 8 h after the ferrous sulfate). Fecal samples were bulked, freeze-dried, and ground to a fine powder. Subsamples were ashed at 480 OC for 48 h in silica crucibles. Ash (0.2 g) was dissolved in 2 mL of warm 6 M HCl and extracted with 2 mL of diethyl ether (13) (“Analar”,BDH Chemicals Ltd., UK). The ether layer was pipetted and transferred to a 1-mL PTFE vial and evaporated to dryness in a stream of air. The residue was redissolved in 20 pL of 1 N HC1. The concentration of iron impurity in these small quantities of reagents was low enough (
