The Rainbow Monitor has proven to be as accurate as, and in some cases, more accurate than, UV and HPLC methods.  The Rainbow approach completely eliminates all errors associated with the removal of samples from the vessels.  These errors can include sample carryover, leaking and/or clogging of transfer lines, pump failures, clogging of filters, absorption of drug onto filters and/or tubing, etc.  With these errors removed, the accuracy and precision of the system is limited by the spectroscopic technique that is employed to make the analytical measurement.

The single largest obstacle to an accurate in situ UV absorbance measurement is interference from excipients.

When the pharmaceutical dosage form dissolves, excipients are released in the dissolution media.

When the pharmaceutical dosage form dissolves, excipients are released in the dissolution media.  Since they are not dissolved in the media, the small particles of excipients are dispersed throughout the media in the dissolution vessel creating a suspension, and the media has a “milky” look.

Molecular absorption from pharmaceutical excipients is rarely a problem if spectral measurements are made at wavelengths longer than 240 nm.  The UV measurements that are made with this system focus on the π (pi) transition that typically occurs between 240 nm and 300 nm.  Since most pharmaceutical active ingredients have an aromatic system, this absorption is very useful for quantitative measurements.  By focusing on this region, we gain a good deal of specificity, since almost all pharmaceutical excipients have no significant UV absorption in this spectral region.  This provides a useful “spectral window” above 240 nm.

Scattering interference by pharmaceutical excipients is typically present at ALL wavelengths across the UV spectrum.  This scattering interference is usually present in one of two forms.  The first is a relative mold scattering that is caused by the film coating of non-disintegrating pharmaceutical dosages.  This form of scattering is evident as a slight (0.01 - 0.1 AU) baseline offset.  The Rainbow Dissolution SmartWare® baseline correction algorithm automatically corrects this type of scattering.  This algorithm simply subtracts the baseline absorbance at a given wavelength from the peak absorbance measurement.  This form of correction works well with non-disintegrating, and even with some disintegrating formulations.

Baseline correction method for a 12-hour dissolution sample:

The second form of scattering interference is much more severe.  This sort of scattering is know as Tyndall scattering (named after British physicist John Tyndall {1820 - 1893}), and is observed in colloidal suspensions.  This sort of scattering arises from the heavy particulate in a disintegrating formulation, or from gelatin capsules.  Gelatin capsules exhibit a severe level of scattering.  This type of scattering produces a sloping baseline, where the interference becomes greater at shorter wavelengths.

Gelatin capsule interference:  Standard spectra (Graph A) without sloping baseline and 24-hour dissolution sample spectra (Graph B) with sloping baseline interference.

Graph A

Graph B

Not only is there a baseline offset, but also the baseline slopes upward from longer to shorter wavelengths.  Additionally, the degree of interference can vary in intensity, as shown by the difference in the baseline for all three samples.  This type of scattering makes it nearly impossible to correct by simply subtracting a placebo spectrum.

The solution to the problem presented in the Figure above is to remove the sloping baseline.  The easiest way to do this is to take a second derivative of each spectrum.  This will serve to remove any sloping offset, since the second derivative of a sloping line is zero.  Here is the example of this, which shows two absorbance scans of caffeine, when scattering correction has occurred.

Correcting for scattering with the second derivative calculation:

Absorbance Spectra-Caffeine

Second Derivative Spectra-Caffeine

The top figure shows an absorbance of the pure caffeine standard, and the same amount of standard with a scattering interference (turbidity standard).  The turbid solution demonstrates a typical turbid interference, with a sloping baseline.  The correction for this interference is illustrated in the bottom figure. It is clearly evident that the second derivative removes the baseline offset and slope of the turbid interference.  The second derivative spectrum of the turbid solution is almost identical to that of the pure standard.  In the wavelength range of 275 - 305 nm, an almost perfect scattering correction has occurred. 

By utilizing these two correction methods (baseline and second derivative), the Rainbow Dynamic Dissolution Monitor® system has been shown to accurately measure dissolution rates of solid forms which produced extremely cloudy solutions.