Introduction

Dissolution is a standard test for evaluating pharmaceutical solid dosage forms.  Dissolution testing (DT) serves as a measure of quality control and sometimes as a means to correlate in-vitro with in-vivo drug release characteristics.

Current United States Pharmacopoeia (USP) dissolution methods most commonly employ a temperature programmable dissolution apparatus (water bath or bathless dissolution), maintained at 37 ±0.5° C, in which sample vessels are placed in a water bath.  In bathless apparatus, sample vessels are heated by external solid state heaters.

The vessels contain a predetermined volume of dissolution media (DM) and a means by which to agitate vessel contents.  This media agitation may be accomplished by a rotating basket attached to a shaft (UPS apparatus 1) or with a paddle which is also attached to a shaft (UPS apparatus 2).  USP has recently introduced other devices and apparatus, but current standard techniques have remained basically unchanged through the last decades.  The solid dosage form is placed into a vessel filled with DM at time zero, and the specific vessel temperature and mixing speeds are maintained.  At allotted times a small sample is removed from each vessel, usually by a multi-channel pumping system, and transported to either a cuvette or a sample vial for subsequent high-performance liquid chromatography (HPLC) or spectrophotometric analysis. 

Of the two methods, HPLC is usually favored and more commonly employed.  HPLC offers the advantage of high specificity as well as acceptable accuracy, precision, and sensitivity, provided that the analytical method has been completely developed and has met strict validation requirements.  Some disadvantages of HPLC are that its labor-intensiveness requires higher skilled personnel, is expensive, and time consuming, and not very productive.

Additionally, the limited number of data points that can be determined using the current HPLC systems may result in a less than ideal representation of the release profile of a solid dosage form over time.  Furthermore, HPLC analysis is a sequential, time-consuming process involving complicated media and sample preparation, system equilibration and standardization, washing of columns and sample-taking system between sample runs.  In general, a typical 24-hour dissolution requires up to 36 hours generating a dissolution profile.

The only substantial modernization of dissolution technology introduced recently was the employment of some elements of robotics in sample taking for testing and some computer software elements for test result calculation.  Neither represents a significant improvement to testing technology that would simplify testing procedures and improve data quality.

Currently used dissolution test methods

Pharmaceutical dissolution testing has traditionally been a labor-intensive process.  Lab technicians must manually drop the tablets, remove several aliquots of solution at specified time intervals, and subsequently analyze the drug concentration in each collected sample.

There are four basic means of sampling from dissolution vessels.  These methods are:  manual sampling (syringe method), sipping to a flow cell (UV only), semi-automatic collection into HPLC vials, and full automatic collection into HPLC vials.  These techniques require volume corrections.

Even with a simple UV absorbance analytical method, a single dissolution run could require an entire day to complete.  Moreover, the time for the DT increases substantially when formulations with multiple components require HPLC analysis.

Dissolution bath/manual sampling method

For DT, a temperature-programmable dissolution bath is usually employed. Sample vessels containing a predetermined volume of DM are placed in the bath along with a means to agitate vessel contents.

The manual sampling method consists of an analyst using a syringe to sample from each vessel at selected time points.  This method suffers from operator-related errors, which vary from analyst to analyst.  In addition, the analyst must remain in the laboratory during the test to conduct the sampling at the set time points.

UV sipper method

The UV sipper method consists of a computer-controlled pump mechanism, which draws an aliquot from each vessel at set time points, directs it to a flow cell situated in a standard bench UV Spectrophotometer (which scans the sample) and then returns the sample to the dissolution vessel.  This approach suffers from a myriad of problems such as pump failures, tubing leaks, clogging of tubing, drug absorption to tubing, etc.

HPLC method

At fixed time intervals (e.g. 2, 4, 8 hours, etc.) a small sample aliquot is taken from each vessel, either manually or by a semi-automatic or automatic device, usually a multi-channel pumping system, and delivered to either a cuvette or a sample vial for subsequent HPLC or spectrophotometric analysis.  Of the two methods, HPLC is usually favored and more commonly employed.  This method separates chemical compounds passing through a column containing active phase via absorption, portioning or other mechanisms.  The separated compounds can then be individually analyzed by a detector and quantitated.  By determining the concentration of analyte in the test media, the percentage of drug dissolved at a given time period can be calculated.  Plotting dissolution of a solid dosage over time results in a dissolution profile.

HPLC offers the advantage of high specificity, as well as acceptable accuracy, precision and sensitivity, provided that the analytical method has been completely developed and has met strict validation requirements.  The disadvantage of HPLC lies with the inherent burden of creating, manipulating, and sorting voluminous numbers of HPLC sequences and data files.  The complexity of sample manipulations is a potential source of errors, or in the worst case, abortion of the analysis.  Multiple manipulations during the testing procedure make HPLC very personnel sensitive, as less skilled lab technicians often incur failures and errors.  The cost of HPLC, columns, mobile phases, and waste solvent disposal, etc is substantial.  Frequent servicing of HPLC instruments is mandatory.

Robotics (semi-automatic and fully automatic HPLC method)

Numerous mechanical sampling systems are available that automate DT with varying degrees of success.  The semi-automatic HPLC method utilizes a robotic sipper, which draws samples from all vessels at set time intervals and places them into HPLC vials located in an external rack.  Once the test procedure is finished, the analyst must place the rack into an HPLC system and begin the assay of the samples. 

The fully automatic collection into HPLC vials consists of a robotic sampler, which like the semi-automatic method, samples all vessels into vials at set time points.  The key difference between the fully automatic method and the semi-automatic method is that the fully automatic method places the samples into vials, which are inside an HPLC system.  Immediately after the samples are drawn from the dissolution vessels, the HPLC analysis begins without any analyst intervention. 

Limitations of traditional dissolution test methods

There are two areas where analytical errors can occur in these traditional methods:  during the sampling procedure, and during the analysis of the samples. 

All four of the above-described methods share one thing in common — they remove a portion of the contents from the dissolution vessel in order to analyze the amount of drug present.  Whenever a liquid is transferred from one container to another, an analytical error can result.  In methods that utilize robotic sampling, the device that conducts the sampling can be either programmed incorrectly or can fail entirely.  Finally, systems that utilize a sipper to transfer sample have a problem with carryover (i.e. contamination of the current sample with residuals from a previous sample).  Although most sampling devices can be programmed to minimize this carryover, this is not usually done. 

In all four of these methods, an inline filter is generally utilized, which can result in the absorption of the active drug onto the filter.  The filters can even become completely clogged, which will result in a missed sampling point.

The UV sipper method has the least effect on the contents of the vessel, since the sample is returned after the analysis.  However, the sample is still removed from the dissolution environment and cooled, which can cause a host of problems. 

One other source of error exclusive to the HPLC analysis (both semi- and fully-automatic) is injector error.  A properly maintained auto injector can be quite reproducible (RSD ~ 0.2% on five injections of standard), but often this level of analytical excellence is not seen.  In fact, an injector RSD of less than 2.0% is considered to be adequate for the analysis of GMP samples.  A variation of 1-2% for five replicate injections (of a standard solution) is a significant error, which is not present in a direct spectroscopic method. 

An additional disadvantage to the HPLC approach is that, following the chromatographic separation step, each component is analyzed by a very crude UV spectrometer.  Most HPLC detectors (UV, single wavelength) have a very wide bandwidth (6-10 nm).  This is purposely done to increase the signal to noise ratio so that the limit of detection can be lowered, but if the chromatographer is not aware of this “feature,” it can cause significant analytical errors.  Beer's law assumes that the radiation interacting with the sample is monochromatic.  As the radiation becomes less monochromatic (bandwidth becomes wider), the deviation from Beer's law will become more significant.  This will be evident in the calibration curve, which will become nonlinear at higher concentrations.  This can make any auto-injector problems much more pronounced if too much sample is injected onto the column (either due to mechanical problem or an improperly programmed system).  What makes this type of error less evident is that most chromatographic data systems report the signal from the detector in millivolt units instead of absorbance units. 

The Rainbow Dynamic Dissolution Monitor® system (UV Fiber Optic Probe Dissolution System) offered by Delphian Technology Inc. is a device that eliminates ALL of the problems previously described (auto injector accuracy, sipper malfunction, carryover, operator error in sampling, filter absorption, volume corrections, poor spectrometer performance).  This system has some unique advantages that will be discussed further.  The removal of these errors is evident in the increased accuracy and precision of this method over current HPLC methods.

The in situ concept has never before been used in dissolution testing.  Its application became possible through the patented invention.  Traditional dissolution testing involves transfer of DM samples outside dissolution vessels for measurement and data generation.  In the Rainbow Dynamic Dissolution Monitor® system, the in situ method reduces dramatically the labor intensiveness of dissolution testing by completely eliminating the need to transfer samples from the test vessel. 

The largest advantage to the in situ approach, other than an increase in the accuracy and precision, is that the vessel can be analyzed much more frequently.  This greater “data density” over time makes the data much more statistically sound.  For a 24-hour dissolution 145 sampling points are taken vs. the usual 3-5 points (once very 10 minutes), which provides a much better statistical base for any data analysis.  Some examples of analysis that are enhanced are in vivo/in vitro correlation, curve predictions, and curve equivalence calculations.  In addition, if an analytical error were to occur at a single datum point, the entire experiment is successful due to the large number of sampling points. 

An additional feature of the in situ approach is that since the analytical procedure is completely electronic, it can be automated.  Furthermore, the lack of moving parts means that the system requires little maintenance and repair.  In addition, the computer is programmed to perform all of the calculations in real time, and generate the dissolution curve as sampling occurs.  This allows the analyst to see how the dissolution is proceeding during the run. In contrast, conventional external techniques perform all of the calculations after the dissolution experiment has been completed. 

UV spectroscopy, which employs the measurement and quantitation of UV absorption by an analyte, is a proven technique.  The use of UV-probes allows measurements in real-time, in the test vessel, without transferring DM samples outside the vessel for testing.  The method significantly reduces the time that an analyst needs to spend with test instrumentation.  Once the testing process is initiated (system calibration, sample and media preparation, etc.) and the sample is deployed into the test media, test data is obtained by the system automatically, and filed electronically in a database without the analyst’s intervention.  The simultaneous use of multiple spectrometers designated to individual dissolution vessels is another innovation in the Rainbow Monitor.

The Rainbow Monitor utilizes high quality spectrophotometers.  The recent model Rainbow Type IIA utilizes six Zeiss PDA detectors, which scan an entire spectrum during the sampling procedure.  Most HPLC-Dissolution systems (and some UV-sipper systems) utilize only single wavelength detection.  The acquisition of a complete scan during the sampling allows for a much more detailed interpretation of the data.  In addition, with the acquisition of a complete scan, many interferences can be eliminated by means of simple (baseline) correction, or more advanced multivariate techniques (second derivative, multiple linear regression principle (MLR),  component analysis (PCA), etc.). 

Fiber optic cables carry monochromatic light signals generated by the UV-probe, in the test media, to a spectrometer, which analyzes the signals and generates test data.  Test data can be generated according to test requirements, and electronically stored.  Fiber optic cables are used in UV/VIS analysis including those related to dissolution; but no one before was able to perform continuous dissolution testing without removing the fiber optics with a probe from the tested media.  Delphian puts the fiber optic cable in the tested media to allow the probe to be constantly submerged in the media, in a dissolution vessel, during the entire duration of a dissolution run.  When necessary, probes can be partially withdrawn from the media by using a manifold.

Our proprietary software package — Rainbow Dissolution SmartWare® was also developed to generate and manage test data without the intervention of an analyst.  This significantly decreases the labor intensiveness and increases data security and reliability in comparison with all currently employed methods for dissolution testing.

The Rainbow Dynamic Dissolution Monitor® system represents a dramatically improved technology for continuously measuring the release of a drug from a pharmaceutical dosage form.  The Rainbow can monitor a single vessel or multiple vessels containing DM and a measuring device for detecting the amount of drug released for a given period of time.  A fiber optic cable can be introduced in the dissolution vessel or placed within the mixing shaft to ensure uninterrupted and continuous data collection by a UV probe during the testing process.  A UV probe is connected to the end of a fiber optic cable emerging from the lower end of the mixing shaft.  Deuterium lamps are used to provide the UV radiation for the analysis.  UV is transmitted from the deuterium lamp by sheathed fiber optic cable to a probe.  In a probe, light travels through a quartz lens seated directly above the flow cell.  UV radiation travels through the flow cell (filled with test media) and reflects off a mirror positioned at the terminal end of the probe.  UV radiation then travels back through the flow cell and quartz lens.  It is detected in a second optic fiber where it travels to the spectrometer for analysis.  Quantification of the drug substance is accomplished by determining the change in UV radiation intensity as it is transmitted through the flow cell.  A spectrometer is integrated with a data processor where data is analyzed in the time intervals required by the experiment and pre-programmed in the system by the analyst.  Data generated by a data processor can be accessed in real time, printed out, electronically filed on a disc or in a secure database, or made available to multiple end-users through network connections.

Computerized data processing and Digital data

All data collected during DT is in electronic form, which can be processed by a computer without any manual calculations.

The digital test data eliminates the need for manual processing of the test result (a routine, time-consuming procedure for HPLC) and allows the data to be handled with a very high level of versatility and security.  The system is designed so that a laboratory technician does not need to process data nor have access to test data until it is finally computed and electronically recorded.

Secured database capability

The system also provides a capability to directly record DT data into a secure database.  Data in such database could be made accessible for manufacturing personnel and for regulatory authorities for test data reference check-up during product shelf life.  This capability dramatically reduces the probability of human error affecting the test data.

The data is stored in a “READ ONLY” manner to allow the data to be viewed, printed, graphed, and exported by the user and to prevent the data from being altered or damaged.  Every spectrum collected on the instrument can be displayed on demand.  Software provides for a secure login to prevent unauthorized users from accessing the program.  The system also has a master event log, which records all actions that occur on the system.  The log is secured by the administrator to prevent the user from changing it.  The software package also allows files to be stored, saving all of the parameters associated with the method.  These method files are secure, and once written, cannot be changed.  All reports generated by the system indicate which method file was used.

Data acquisition frequency and consistency

The system allows collection and computation of test data as often as required, not only every 2, 4, 6 hours, etc. as usually done, but in increments of minutes, and even seconds.  This capability allows absorption dynamics data to be obtained for quickly dissolving (absorbed) medications, as well as for those medications that contain very small amounts of active substance (few milligrams).

Additionally, the system allows absorption dynamics to be viewed in a 3-D form (dissolution time vs. absorbance vs. wavelength).  The system is able to perform real-time calculations on all data.  The required calculations are % dissolved and system suitability.  The % dissolved is calculated in one of two ways, either a two-wavelength (analytical wavelength, and baseline correction) or a three-wavelength method.

Economics

In addition to the above-described advantages, the new dissolution system allows customers to save money on laboratory consumables, waste management, and technician’s labor.  The new system does not require any additional solvents or specialty chemicals for operation.  This means that operating the system generates no chemical waste.

One lab technician will spend 1.25 hours for one dissolution test with the Rainbow NCD system versus 6 hours with HPLC.

The tables below are presented to demonstrate the particular features of the Rainbow Dynamic Dissolution Monitor® system versus HPLC.

Time requirement for a dissolution test:

HPLC		RAINBOW Monitor
Function	Time Required to Perform Function (hr)		Function
Experiment	24	24	Experiment
Pre-experiment setup	1	0.5	Dissolution mobile phase preparation/ Documentation/ Autosampler setup/Pump setup/ Chromatography acquisition setup
Equilibration	1	0	System Equilibration N/A
System Suitability	1	0.2	System Suitability
Reintegration	1	0	Post Run Data Processing N/A
Post Run Calculations	1	0	Manual Calculations / Report Generation N/A
Clean up	1	0.5	Post Run Clean up / Report Generation
Total Time for Experiment	30	25.2	Total Time for Experiment
Total Analyst Time	6	1.2	Total Analyst Time

Annual operation cost of HPLC vs. Rainbow Monitor:

HPLC		RAINBOW Monitor
Item	Cost		Item
1 HPLC Column	$250	$395	1 Deuterium Lamp (2000 hrs)
5 Guard Columns	$250	0	N/A
1 Major Repair	$1000	0	N/A
Vials	$300	0	N/A
Seals/Tubing, etc.	$300	0	N/A
Solvents	$400	0	N/A
Waste Disposal	Variable on Quantity	0	N/A
Total Annual Expense	$2500 + Waste Disposal Cost	$395	Total Annual Expense

Delphian Technology will be delighted to share our knowledge of the fiber optic dissolution with our distinguished colleagues across the pharmaceutical industry.

See for yourself what the Rainbow Monitor can do for you at your laboratory.