Noel M. Meltzer

Dermal Microdialysis Sampling in Vivo

Microdialysis sampling of the dermis in vivo was accomplished using a linear microdialysis probe. In contrast to previous studies using a commercial cannula-style microdialysis probe, the linear probe had no effect on the flux of drug through the skin in vitro. The extent of tissue damage in vivo due to probe implantation was evaluated by histological examination and microdialysis delivery studies. Tissue damage due to implantation of the linear probe was minimal with no bleeding or edema observed. Infiltration of lymphocytes into the tissue was observed beginning 6 hours after probe implantation with scar tissue beginning to form after approximately 32 hours. The infiltration of lymphocytes had no effect on the behavior of implanted microdialysis probes. Delivery of 5-fluorouracil was between 20 and 25% for six different probes implanted in six different animals demonstrating good probe-to-probe and implantation-to-implantation reproducibility. Constant delivery was maintained for at least 24 hours in all cases indicating that experiments of at least 24 hour duration are feasible. The dermal concentration of topically applied 5-FU cream, Efudex®, was continuously monitored by an implanted microdialysis probe demonstrating the feasibility of this technique as for monitoring skin drug levels in vivo. The dermal concentration of 5-FU following topical application was approximately 40-fold higher for in vitro excised skin than for in vivo intact skin.

Microdialysis sampling for the investigation of dermal drug transport

Microdialysis perfusion in vivo has the potential to be a powerful sampling technique in dermal and transdermal drug delivery studies. Characterization of a commercially available microdialysis probe in vitro considering relevant physiological parameters is a vital first step in the evaluation of microdialysis as a dermal sampling technique. In previous microdialysis studies, analyte concentration and neutrality have been implicated in altering microdialysis recovery. The recovery of a model compound 5-fluorouracil (5-FU) was investigated at several pH values and donor concentrations. The relative recovery of 5-FU by the microdialysis probe was affected by pH but not by donor concentration. To confirm further that the changing concentration and pH profile presented by the flux of 5-FU was not significantly altering microdialysis recovery, an experiment comparing direct and microdialysis sampling of a Franz diffusion cell receptor compartment was performed. Although the 5-FU concentration (0-686 ng/ml) and pH (7.40-7.24) changed substantially, the recovery of 5-FU was not adversely affected. To demonstrate the feasibility of dermal microdialysis, the flux of a commercial preparation of 5-fluorouracil was monitored utilizing a microdialysis probe implanted in excised rat skin in vitro. The results from the dermally implanted probe demonstrate the potential of the technique while establishing the limitations of the current microdialysis system.

Normal phase LC-MS determination of retinoic acid degradation products.

The degradation products formed when 13-cis retinoic acid (13-cis RA) and all-trans RA were exposed to fluorescent light and air were investigated. These retinoids are known to undergo Z-E isomerization (due to the existence of four unsaturated double bonds) and oxidation when exposed to light and air. Analysis by LC was carried out on a 25 cm x 4.6 mm Zorbax Rx-SIL (5 microns) with a mobile phase (1.4 ml min-1) of heptane-THF-acetic acid (96.5:3.5:0.015) and an in-line UV (365 nm) detector. The LC eluate was coupled through a Vestec universal interface to a Finnigan 4023 mass spectrometer. EI-mass spectra were obtained at 77 eV from m/z 200 to 350 with multiplier voltage of 1200 V. Solid samples of 13-cis RA and all-trans RA exposed to light and air and also solutions of these retinoids in the mobile phase exposed to the same conditions were used for the analysis. Tentative identities of the degradation products from the mass spectra suggest the isomerization of the retinoids (Z-E isomerism) and the formation of the 5,6-epoxides of these isomers. Identities of the 5,6-epoxides were confirmed with chromatographic and mass spectral data from synthetic samples of the epoxides. Isomerization occurred more readily in solution than in the solid form and the 13-cis RA isomer oxidized more readily than the all-trans isomer.

Solid-state stability studies of 13-cis-retinoic acid and all-trans-retinoic acid using microcalorimetry and HPLC analysis

The solid-state stabilities of 13-cis-retinoic acid and all-trans-retinoic acid in the presence and absence of oxygen were investigated. The samples were first evaluated using microcalorimetry. The rate laws of different samples under different conditions were deduced from the shapes of the heat flow curves, and the activation energies of the reactions were determined from Arrhenius plots. Under an air atmosphere, the decomposition of 13-cis-retinoic acid is an autocatalytic reaction, while all-trans-retinoic acid undergoes a zero-order process. The degradation of the two compounds at a selected elevated temperature was also determined utilizing HPLC analysis. This technique confirmed the decomposition kinetics. Hence, their half-lives and shelf lives at room temperature could be calculated. Under a nitrogen atmosphere, the microcalorimetric experiment showed a first-order phenomenon for both samples, but HPLC analysis showed no degradation, suggesting that the two samples, in the absence of oxygen, undergo only a physical change.

Separation of 13-cis and all-trans retinoic acid and their photodegradation products using capillary zone electrophoresis and micellar electrokinetic chromatography (MEC)☆

Two retinoic acid isomers; 13-cis retinoic acid and all-trans retinoic acid and their photodegradation products were resolved with capillary electrophoresis (CE) (UV detector, 345 nm) using three different mobile phases: method 1--an acetonitrile modified borate buffer (pH 8.5); method 2--borate buffer (pH 8.5) modified with acetonitrile and alpha-cyclodextrin; and method 3--borate buffer (pH 8.5) modified with SDS (MEC). Concentration of acetonitrile in the buffer was varied from 10 to 50% in method 1 and resolutions of 0-1.9 were obtained for the two retinoic acid isomers. Similarly in method 2, concentration of alpha-cyclodextrin in the buffer (with 10% acetonitrile) was varied from 0 to 40 mM, giving resolutions of 0-3.8. In method 3, concentration of SDS in the buffer was varied from 5 to 60 mM resulting in resolutions of 1.3-4.1. Optimum separation conditions for the three methods were applied to the separation of photodegradation products of the two retinoids after exposure to fluorescent light for 36 h. A buffer modified with 45% acetonitrile and the same buffer modified with 10 mM SDS gave incompletely resolved electropherograms with a 72 cm x 50 microns capillary (50 cm to the detector). A buffer containing 20 mM alpha-cyclodextrin 10% acetonitrile gave completely resolved peaks for each isomer. The buffer containing 10 mM SDS gave completely resolved peaks for the photodegradation products when a 122 cm x 50 microns capillary (100 cm to detector) was used.

Determination of the kinetics of degradation of 13-cis-retinoic acid and all-trans-retinoic acid in solution☆

The degradations of 13-cis-retinoic acid and all-trans-retinoic acid in an organic solvent were determined with an HPLC assay. The degradation curves at 70, 50 and 37 degrees C all showed autocatalytic characteristics for both isomers. For this kind of complex reaction, the usual method cannot be used to estimate the shelf-lives and half-lives at room temperature. In this work a new method was developed to directly calculate the shelf-lives and half-lives. From this equation the activation energy was found to change as the multiple step reaction progressed.

Development and Validation of Method for Determination of In Vitro Release of Retinoic Acid from Creams

AbstractThe objective of this study was to develop and validate an in vitro test method that can be used as a tool for accessing batch-to-batch uniformity of finished topical products. The studies were performed by utilizing topical creams containing the 13-cis isomer of retinoic acid. Various physicochemical factors which may affect drug release from topical cream formulations were evaluated including drug concentration, internal phase droplet size distribution, viscosity and the composition of the emulsion internal and external phases. Utilizing a modified Franz diffusion cell with a cellulose membrane, the in vitro drug release profile from various cream formulations was establishedThe effect of varying the concentrations of various key ingredients by 30% does affect the viscosities and release rates compared to a standard formulation. However, there is no correlation between the viscosities and the release rates. No significant differences in pH and droplet size distribution were observed in these cre...

Electrochemical detection of retinoids using normal phase HPLC

Abstract Non-aqueous electrochemical (EC) detection of 13-cis-retinoic acid, all-trans retinoic, acitretin and vitamin A palmitate in non-aqueous solvents are reported. Non-aqueous (EC) detection allows for normal-phase chromato-graphy of these compounds prior to detection. The normal phase system used a mobile phase of HEX/THF/AcOH for the separation of all four compounds. The stationary phase was either silica or PVA-sil. The lipophilic salts, t-butylammoniumtetrafluoroborate or t-butyl-ammoniumhexafluorophosphate necessary for EC detection were added post-column. The limit of detection (LOD) for EC detection of these compounds is approximately 1 ng on column compared with an LOD by UV absorption of 2 ng on column. The linear detection for these compounds with the EC detector was about two orders of magnitude.

Normal Phase HPLC with EC Detection: 1,25-Dihydroxy Vitamin D3 and 25-Hydroxy-16-ene-23-yne Vitamin D3

Abstract An analytical method for the separation and detection of 1,25-dihydroxy vitamin D3 and 25-hydroxy-16-ene-23-yne vitamin D3 by normal phase HPLC with electrochemical detector is reported. A post column electrolyte technique was adopted in order to apply electrochemical detection in a normal phase chromatographic system. Warfarin was used as an internal standard. The response and concentration was linear over the 10−3 mg/ml−10−5 mg/ml concentration range for both compounds. The sensitivity is improved 1000 fold by the use of amperometric detection instead of UV detection. The detection limit was approximately 3 pg/ml. The amperometric detector can detect some of the degradation products of 25-hydroxy-16-ene-23-yne vitamin D3 which can not be detected by UV.