No abstract

With capillary electrophoresis, buffer pH must be constant to achieve consistent migration times. Irreproducible separations have been attributed to pH changes due to water hydrolysis in the inlet/outlet vials. A method of measuring the pH of the electrolyte on-column is described that uses wavelength-resolved fluorescence detection. C.SNARF-1 is a fluorescent pH indicator that has a large change in fluorescence emission profile depending on pH. When it is incorporated into the running buffer, monitoring the pH-dependent emission spectra of the C.SNARF-1 allows column pH to be calculated. With reduced-volume outlet buffer vials in the nanoliter to low microliter range, significant changes in pH and column conductivity are measured during a single electrophoretic run, with pH fronts greater than 3 units passing a fixed point on the capillary over a several second period. These changes appear to be caused by reverse-migrating OH- produced at the capillary outlet by the hydrolysis of water.

A postcolumn radionuclide detection system for capillary electrophoresis (CE) is described. Eluant from an electrophoresis capillary is directed onto a peptide binding membrane that has been previously coated with a solid scintillator. The membrane is moved in a preselected pattern relative to the fixed capillary outlet during electrophoresis. Light emission from scintillation is imaged onto a charge-coupled device (CCD) using a series of 35-mm camera lenses. Detection of two low-energy beta- emitters (35S and 3H) not previously reported for capillary electrophoresis is demonstrated. The separation efficiencies are similar to those obtained with on-line UV detection. The response for 35S-labeled methionine is linear (r2 = 0.996) from 66 amol to 11 fmol. Detection limits are 88 zmol (0.03 Bq) for 32P-labeled analytes, 17 amol (0.94 Bq) for 35S-labeled analytes, and 8 fmol (8.5 Bq) for 3H-labeled analytes.

A capillary electrophoresis (CE) postcolumn radionuclide detector has been developed that uses a commercial phosphor-imaging detector and has been optimized for low-energy beta emitters. Eluant from the separation capillary is deposited on a membrane. Emission from radioactive analytes on the membrane is integrated using the phosphor-imaging system for 10-72 h. Results from the phosphor-imaging system are converted to conventional electropherograms. Modifications to a prior postcolumn CE deposition system have been accomplished by adding a buffer makeup capillary; this increases the electrolyte pH range and improves reproducibility. The limit of detection (LOD) for 35S-labeled analytes is 0.13 amol (8.7 pM or 0.007 Bq), while the LOD for 32P-labeled analytes is 4.9 zmol (0.33 pM or 0.002 Bq), with a linear range for 35S-Met from 1.5 amol to 1.5 fmol.

The stability of the 37− amino acid peptide pramlintide, in aqueous solution, was studied as a function of pH and temperature. Samples of pramlintide formulated as a parenteral product were exposed to elevated temperatures and to realistic storage conditions for as long as 30 months. Pramlintide degradation was monitored by three high-performance liquid chromatography (HPLC) methods: a reversedphase (RP-HPLC) and a strong-cation exchange (SCX-HPLC) method for percentage purity determination by area normalization, plus a second RP-HPLC method for potency determination versus external standards. The pH-rate profile for pramlintide shows increasing degradation rate constants with increasing pH over the range pH = 3.5 to 5.0. The Arrhenius expression for pramlintide degradation at pH = 4.0 over the temperature range 5°C to 50° C is ln(k 0)= 37.39-21.900/RT, where k 0 is the zero-order rate constant (in %/mo) for pramlintide degradation. The pramlintide parenteral product formulated at pH = 4.0 is extremely stable, with percentage purity and percentage potency loss of only approximately 2% over 30 months at 5°C. The formulated pramlintide drug product has acceptable shelf life for long-term storage at 5°C and up to a 30-day patient use when stored at ambient temperature.