A number of biological reference materials (RM) have been prepared in our laboratory specifically for validating analytical methods for the determination of Pb in biological matrices (e.g. blood, urine, liver, and bone). The RM's were developed using animal (goats and cows) that are routinely dosed with lead acetate to produce proficiency test samples for blood lead (and erythrocyte protoporphyrin). In cases where an animal becomes injured or infirm, the veterinarian in charge may recommend that the animal be euthanized. In such cases, samples of bone, brain, liver, and other tissues containing lead are removed at autopsy.
Currently, we have collected bone samples from nine goats and one cow that were dosed with lead over periods ranging from 1 to 10 years, During the autopsy, the epiphyses (bone joints) are separated from each long bone. Skin, muscle, and other adhering tissues are dissected or scraped from each bone. Bone marrow is also removed. All bare bones are currently stored at -70°C until analyses for Pb are conducted.
The only certified reference materials for bone Pb are those available from the National Institute for Standards and Technology (NIST), Gaithersburg, MD. Standard Reference Material (SRM) 1486 Bone Meal has a certified Pb concentration of only 1.335 μg/g. This is close to normal for humans, but is too low to be of practical use for in vivo X-Ray Fluorescence (XRF) equipment, SRM 1400 Bone Ash has a certified Pb concentration of 9.07 μg/g. Neither SRM is optimal for validating in vivo XRF equipment, but they are both very useful in validating other analytical methods for bone Pb such as Graphite Furnace Atomic Absorption Spectrometry (GFAAS).
We have developed an accurate, precise, and sensitive method for determining Pb in bone using GFAAS with Zeeman background correction. Using this method, we have analyzed the animal bones for Pb. Bone samples were divided into smaller pieces using a diamond-disc saw, freeze dried, and homogenized in a tantalum ball mill. Samples of bone powder were digested in nitric acid using a closed vessel microwave digestion system. Lead was determined using aqueous Pb standards in a chemical modifier optimized for the bone matrix. The method was validated using NIST SRM Bone Meal and Bone Ash. The detection limit is 0.6 μg/g based on 3 SD. Results for Pb in our animal bone range from approximately 5 to 50 μg/g dry weight. The results indicate that the intact bare bones would be excellent candidates for interlaboratory studies of in vivo XRF measurements of bone Pb. They are stable, well-characterized, easily transported between sites, and cover the clinically relevant range of bone lead concentrations likely to be encountered in the field. It is proposed that these materials be circulated as part of an interlaboratory comparison to interested centers using in vivo XRF After the XRF analyses, the bone samples will be analyzed for Pb by GFAAS for comparison purposes.