Previously we obtained number average molecular weight (Mn) data for Dextran 70 and Dextran 40 (colloidal plasma volume expanders) by membrane osmometry. These values demonstrated that the HPLC-gel permeation chromatography (GPC) methodology in the proposed USP Monographs for the Dextrans can give correct Mn values as well as weight average molecular weight (Mw) values, for which the method was originally proposed. The ratio Mw/Mn, which is greater than or equal to 1, affords an assessment of the polydispersity of the material, with unity indicating a monodisperse preparation. Membrane osmometry has also permitted an estimate of the upper limit of Mn for Hetastarch (another colloidal volume expander), <46,700>5; only an upper limit could be measured due to the presence of low concns of lower molecular weight species that were membrane permeable because although Hetastarch is substantially larger than either Dextran, it is much more polydisperse. This upper limit has proved extremely important in resolving the conflict among Mn values being reported for Hetastarch by manufacturers applying for approval of a generic Hetastarch and being reported by the innovator itself; all such reported Mn values derive from some sort of HPLC-GPC method. A single manufacturer, using HPLC-GPC, reports Mn values that are in accord with the upper limit determined by membrane osmometry. At the request of CBER, the Division of Drug Analysis of CDER has duplicated the HPLC method of this manufacturer and analyzed a number of samples of Hetastarch obtaining both Mw and Mn values for each. In a collaboration with Dr. R. Ghirlando of NIH, we are now determining Mw values for some of these samples by light scattering in order to obtain an independent confirmation that the HPLC method gives correct Mw values also. The light scattering instrument uses an argon laser (lambda = 514 nm), and measurements are extrapolated to zero concn and zero angle by means of a Zimm plot. We have determined (dn/dc)lambda = 546 for Hetastarch and use it to determine Hetastarch concn. Preliminary results (i.e. uncorrected for small mismatches in NaCl concn) indicate agreement between the HPLC and light scattering methods thereby validating this particular HPLC procedure.
