Method for enantiomeric purity of a quinuclidine candidate drug by capillary electrophoresis.

A chiral procedure based on EKC was developed and validated for determination of the enantiomeric purity of PHA-543613, a drug candidate that was under development for treatment of the cognitive deficits of Alzheimer's disease and schizophrenia. Separation of enantiomers is accomplished via differential, enantiospecific complexation with a single-isomer, precisely sulfated beta-CD and heptakis-6-sulfato-beta-CD (HpS-beta-CD). Both neutral and sulfated CDs were screened before selecting HpS-beta-CD as the chiral selector. The separation is conducted in a 61 cm x 50 microm uncoated fused silica capillary with 25 mM HpS-beta-CD in pH 2.50, 25 mM lithium phosphate as the separation buffer with detection at 220 nm. Application of reverse polarity at -30 kV results in an elution time of about 12 min for PHA-543613 and 13 min for the undesired S-enantiomer. Quantification is versus an authentic reference S-enantiomer as an external standard in combination with an internal standard. The procedure was validated over the range 0.1-2.0% w/w. The detection limit is 0.01-0.02%. The amount of distomer intrinsic to the drug substance is about 0.1% or less. The developed method was used to generate stability data on multiple lots: in one case for up to 3 years.

Enantiomeric purity methods for three pharmaceutical compounds by electrokinetic capillary chromatography utilizing highly sulfated-gamma-cyclodextrin as the chiral selector.

Methods for enantiomeric purity by electrokinetic chromatography were developed and validated for three pharmaceutical compounds, each utilizing highly sulfated-gamma-cyclodextrin (HS-gamma-CD) as the chiral recognition agent. Two of the compounds are weak bases, hence charged at low pH, and the third is a quaternary nitrogen compound, charged at all pH. In each instance quantification was via an authentic reference standard with addition of an internal standard. Separation was on a 61 cm x 50 microm untreated capillary under reverse polarity with a background electrolyte of 5% HS-gamma-CD in pH 2.50 lithium phosphate buffer. Each method was validated with respect to the usual validation parameters, notably recovery and precision, yielding results, including limits of detection and quantitation, that allow reporting the minor enantiomer to 0.1% and less. In applying the methods, all batches of bulk drug tested were shown to be of enantiomeric purity > or =99.9%.

Determination of the molecular complexation constant between alprostadil and alpha-cyclodextrin by conductometry: implications for a freeze-dried formulation.

The binding constant between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD) was determined at four temperatures using conductance measurements. Alpha-cyclodextrin is an excipient material in Caverject dual chamber syringe (DCS) that was added to enhance stability. The binding constant was used to calculate the amount of PGE1 free upon reconstitution and injection, since only the free drug is clinically active. The conductivity measurement is based on a decrease in specific conductance as alprostadil is titrated with alpha-CD. The change in conductivity was plotted versus free ligand concentration (alpha-CD) to generate a binding curve. As the value of the binding constant proved to be dependent on substrate concentration, it is really a pseudo binding constant. A value of 742+/-60 M(-1) was obtained for a 0.5 mM solution of alprostadil at 27 degrees C and a value of 550+/-52 M(-1) at 37 degrees C. These results compare favorably to values previously obtained by NMR and capillary electrophoresis. Calculation of the fraction PGE1 free upon reconstitution and injection show it to approach the desired outcome of one. Hence, the amount of drug delivered by Caverject DCS is nominally equivalent to that delivered by Caverject S. Po., a predecessor product that contains no alpha-cyclodextrin.

A method for the determination of minoxidil in hair-regrowth formulations by micellar electrokinetic capillary chromatography.

A method based on micellar electrokinetic capillary chromatography (MEKC) was developed for determination of minoxidil in Rogaine and competing products. The original intent of the work was to offer an orthogonal means to HPLC for testing illicit imitations of Rogaine. However, because the patent has since expired, we offer the procedure as a confirmatory measure to HPLC for assay of generic minoxidil products. The MEKC procedure complements an earlier method based on free solution capillary electrophoresis (FSCE), designed to the same end. Validation was carried out on both a Dionex CES-1, which utilizes gravity injection, and a PE-ABI 270HT, which employs vacuum injection. The procedure was validated for both active pharmaceutical ingredient and for minoxidil solutions. The run buffer is pH 7.0, 20 mM sodium phosphate, 20 mM sodium dodecyl sulfate, with 10% isopropanol; the internal standard is dl-tryptophan. The method bears the attributes of simplicity, ease of use, and short analysis time (12 min). It is selective with respect to known process and degradation impurities. High efficiency was achieved on the CES-1, with a plate count exceeding 200,000 for minoxidil at an elution time of 9 min. Although slight differences in performance were noted across the two instruments, results on both were in conformance with modern day validation expectations. Comparison of MEKC with HPLC resulted in slightly higher values for the former, but all results met registration specifications and internal targets.

Rapid determination of trace amounts of minoxidil in hamster skin follicles with various formulations using narrow-bore LC/EC.

A sensitive liquid chromatographic method with electrochemistry (LC/EC) was developed for the determination of trace of minoxidil in hamster skin follicles after topical administration of the ear using various formulations. The minoxidil in the sebaceous glands of the hamster ear was isolated from the skin and the follicles in different skin layers were treated with aqueous trichloroacetic acid followed by acetonitrile. The supernatant was directly injected into the LC/EC system and minoxidil was detected by oxidation at +800 mV versus Ag/AgCl using a glassy carbon electrode. The analytical recoveries were between 94.4 and 103.1% and the linearity was excellent up to 250 microg/ml with a regression coefficient (r(2)) of 0.9988. The LC/EC and the widely used radiolabeled scintillation methods agree well and both show high sensitivities. The LC/EC method is rapid and cost-effective with a detection limit of only 1 ng/ml.

Determination of the binding constant between alprostadil and alpha-cyclodextrin by capillary electrophoresis: implications for a freeze-dried formulation.

The binding constant between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD) was determined at three temperatures by capillary electrophoresis. alpha-CD is an excipient material in Caverject Dual Chamber Syringe (DCS), added to enhance stability. The binding constant was used to calculate the amount of PGE1 free upon reconstitution and injection, the latter of which is critical to product performance. Measurement was made in a pH 7.2 separation buffer to ensure a negative charge on PGE1. The concentration of PGE1 was fixed while the concentration of alpha-CD was varied over a suitable range. As the amount of PGE1 bound to alpha-CD increases, the weighted average of the resultant mobility decreases, thereby allowing a binding isotherm to be generated from which the stability constant was extracted via nonlinear regression analysis. A value of 708 +/- 64 M(-1) was obtained at 27 degrees C, while at physiological temperature (37 degrees C) the value was 537 +/- 27 M(-1). These results compare favorably to values previously obtained by NMR. Calculation of the percent PGE1 free upon reconstitution and injection show it to be near the desired outcome of 100%. Hence, we were able to conclude that the amount of free drug delivered by Caverject DCS is nominally the same as for Caverject S. Po., an earlier-developed product that contains no alpha-CD.

Determination by NMR of the binding constant for the molecular complex between alprostadil and alpha-cyclodextrin. Implications for a freeze-dried formulation.

A binding constant was determined for the complexation reaction between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD). This constant was used to calculate the fraction PGE1 free upon reconstitution of Caverject dual chamber syringe, indicated for the treatment of erectile dysfunction. The determination was based on the measurement of the chemical shift of the C20 methyl protons of PGE1. The observed chemical shift varies as a linear function of the amount of PGE1 bound. The binding constant was obtained from the binding isotherm, a curve of the observed chemical shift versus free ligand (alpha-CD) concentration, through the application of non-linear regression analysis. A value K11 = 966 M(-1) +/- 130 M(-1) (2s), measured at 27 degrees C, was obtained. This value is in good agreement with those reported in the literature. The percent PGE1 free was subsequently calculated for the reconstituted solution and in the corpora cavernosum after injection. The latter showed PGE1 to be delivered essentially quantitatively to the targeted site.

Determination of protein-drug binding constants by pressure-assisted capillary electrophoresis (PACE)/frontal analysis (FA).

A simple method for the determination of binding constants of drugs to human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) was developed by pressured-assisted capillary electrophoresis (PACE) based on the principle of frontal analysis (FA). The free drug concentration was measured from the height of the frontal peak and calculated based on the external drug standard in the absence of protein. With a known concentration of total drug, the percentage of drug bound to HSA or AGP was then determined. The binding constants of drug to HSA or AGP were obtained from non-linear curve fitting of the percentage of bound drug as a function of total protein concentration or total drug concentration. The sample was prepared by mixing known concentrations of drug and protein in phosphate buffered saline (PBS) and equilibrated for 30 min. A large volume of sample solution (approximately 80 nl) was injected at 1.0 psi for 40 s into the fused silica capillary, which was filled with PBS buffer. Due to the difference in charge/size ratio, the free drug was separated from the protein/protein-drug complex when 15-25 kV voltage and 0.5-1.5 psi air pressure were applied. External air pressure was used to improve the throughput, prevent protein loss, and achieve a better drug plateau. By modifying experimental conditions, a wide range of binding constants could be measured. This PACE/FA method works well for basic, neutral, and weakly acidic compounds.