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Using the SpectraMax Plus for USP Dissolution Calibration (MaxLine Application Note #27)


INTRODUCTION
Calibration of a dissolution apparatus is necessary to produce accurate results for dissolution testing of pharmaceutical dosage forms and to allow comparison between batches and between laboratories. USP requirements for calibration are given in the Apparatus Suitability Test in USP <711> Dissolution.1 Each individual apparatus (i.e. spindle or basket) must be tested with disintegrating type and non-disintegrating type USP dissolution calibrators. The calibrator tablets available from the USP-NF include prednisone, (50 mg, disintegrating type) and salicylic acid (300 mg, non-disintegrating type). The test involves determining the percentage of drug that dissolves in 900 mL dissolution medium under specified conditions (50 or 100 RPM) after a specified time interval (30 minutes). The apparatus is deemed suitable if the results are within the acceptable range stated in the certificate for the calibrator (i.e the specifications sheet for that particular lot number of calibrator).

USP <711> specifies that both prednisone and salicylic acid are to be measured by UV spectrophotometry (242 nm and 296 nm) by comparison to a standard solution of known concentration. (Compendial requirements for UV spectrophotometry are given in USP <851> Spectrophotometry and Light Scattering.2)

This application note describes how spectrophotometric analyses for dissolution calibration can be quickly and conveniently carried out in a 96-well format using the SPECTRAmax PLUS microplate spectrophotometer. The PathCheck feature of the SPECTRAmax PLUS automatically corrects the results to a 1 cm pathlength, therefore samples read in a microplate give the same values that they would if read in a standard cuvette. Accurate pipetting of the samples into the wells is not necessary; indeed PathCheck corrects for pipetting errors. Using flexible custom formulas and the spreadsheet capability of SOFTmax PR O software, the data are calculated automatically and final results are displayed as overall PASS/FAIL for each apparatus.


MATERIALS

For detailed information on the principles of pathlength correction, refer to MAX-line application note #25: Optical density measurements automatically normalized to a 1 cm pathlength in the SPECTRAmax PLUS microplate spectrophotometer.

1. SPECTRAmax PLUS microplate spectrophotometer

2. High-quality UV transparent microplates; e.g.:
SPECTRAplate-Quartz (Molecular Devices Corp., catalog # R8024)
UV Plate, disposable (Corning Costar Corp., catalog # 3635)

3. Calibrator tablets: disintegrating type (prednisone, 50 mg), and non-disintegrating type (salicylic acid, 300 mg) (USP-NF Reference Standards, Tel: (800) 227-8722)

4. USP prednisone and USP salicylic acid reference standards (USP-NF Reference Standards, Tel: (800) 227-8722)

5. Phosphate buffer, 0.05 M, pH 7.40 + .05 (pH tested at room temperature), for salicylic acid test only (Sigma Chemical Co., ready-to-reconstitute powder; GAL-PAC, catalog #936-4GP)

6. Pipettor and tips or transfer pipets suitable for use with microplates


METHOD

Note: Your standard operating procedure (SOP) should already address your particular apparatus and USP requirements such as instrument variables (e.g. height of paddle or basket in vessel, shaft straightness, vessel centering, deaeration, verification of temperature and spindle speed).

Prepare the samples and standards
Step 1 Set up your dissolution apparatus and conduct the test according to your SOP.

Step 2 Collect the samples at the specified time (typically 30 minutes) and filter them according to your SOP. Refrigerate the samples if they are not to be analyzed immediately. The required sample v olume for each micro-plate well is 100-300 mL.

Step 3 Prepare the standard solutions. For this application note, standards were prepared in concentrations ranging from 5.0 to 50 mg/mL for prednisone and from 7.2 to 72.2 mg/mL for salicylic acid.


Set up for the pre-read measurements

Step 1 Launch SOFTmax PRO, then open a Plate Section or, if necessary, create a new Plate Section.

Step 2 Set up the Instrument Settings dialog box as shown in Figure 1. Select to perform an endpoint read at the desired wavelength (242 nm for prednisone; 296 nm for salicylic acid). Click the PathCheck box to mark it, (Pre-read plate will automatically be checked), then click the Cuvette Reference button. Alternatively, you can select the Water Constant button to use the factory-installed Water Constant instead of a cuvette reference, though it is potentially less accurate.


Step 3 Use the Template Editor to create a template showing where standards, blanks and unknowns will be located on the microplate.

Step 4 Configure the Reduction dialog box as shown in Figure 2. Set the wavelength combination to L1 and the data mode to Absorbance. Make sure that Use pre-read plate and Apply Pathcheck are selected.


Making pre-read measurements

Note: In some cases, you may want to use stored pre-read values (see below).

Step 1 Select a clean microplate and pipet distilled water into each well in approximately the same volumes as the intended samples. Accurate pipetting is not necessary.

Step 2 Put the plate into the SPECTRAmax PLUS drawer, and make the pre-read measurements by clicking the Read button in SOFTmax PROs toolbar.

Step 3 Remove the plate, pour out the water and blot the plate dry.


Making pathl ength-corrected absorbance measurements

Note: if you have elected to use the pre-installed Water Constant, skip Step 1.

Step 1 Place a clean quartz or glass cuvette containing distilled water or your dissolution medium in the cuvette port of the SPECTRAmax PLUS. The cuvette is needed for the 1 cm near infrared (NIR) measurements for pathlength correction. A plastic cuvette may also be suitable, but you should verify that your particular plastic cuvette gives the same path-length correction as a glass or quartz cuvette.

Step 2 Transfer aliquots of your samples, standards, and blanks (if any) into their designated wells in the microplate. Accurate pipetting is not necessary, though the volumes should between 100 L and 300 L for best pathlength-corrected results.

To avoid evaporation error, read the microplate within a few minutes of putting the samples in it. If the readings must be delayed, cover the plate with an adhesive seal. Remove the seal immediately prior to reading the plate.

Step 3 Put the plate into the SPECTRAmax PLUS drawer, then click the Read button in SOFTmax PROs toolbar to make the absorbance measurements. The cuvette will be read at the same time the plate is read, and its NIR absorbance values used in the PathCheck calculations (or, if using the Water Constant, the constant will be used to correct the absorbance values).


Storing and re-using pre-read data
Quartz plates are so uniform that it is not necessary to subtract the plate background on a well-by-well basis. Instead, you can pre-read one plate, then store the pre-read data in a SOFTmax PRO file for subsequent re-use, being careful to save the data under a different file name each time, to avoid over-writing the pre-read data.

If you use Costar UV plates, you may or may not want to use stored pre-read values, depending on the precision you require. At 296 nm, the average absorbance of Costar wells containing water is approximately .039 to .049 OD. Thus, for samples having raw absorbance values of 1 OD, the worst-case mismatch between plates would still produce an error of 1% or less. However, the lower the absorbance values of the samples, the greater the maximum potential error due to plate variability. The variability is considerably higher at 242 nm, where the average absorbance is approximately .090 OD, and the standard deviations range from .002 to .015 OD. If you decide to use Costar UV plates with stored pre-read values, potential error can be minimized by analyzing all samples and standards in duplicate or triplicate.


CALIBRATION TEST OF USP APPARATUS 2
To illustrate the performance of the SPECTRAmax PLUS, two 6-paddle (Apparatus 2) dissolution systems were tested using USP disintegrating (prednisone) calibrator tablets. The tests were performed according to the specifications in USP <711> and the instructions on the certificate accompanying the calibrator. The absorbances of the standards and samples were read in a microplate format using SOFTmax PROs PathCheck feature to normalize the absorbance values to a 1 cm pathlength. The resulting data were analyzed using custom formulas written in SOFTmax PRO.

Experimental details
To make the standards, a stock solution of USP Prednisone reference standard was prepared in water containing 5% alcohol. Aliquots of the stock were diluted with water to give final working standard concentrations of 5, 10, 20, 30, 40 and 50 g/mL.

The USP calibrator tablets were Prednisone, 50 mg, Lot L. Two dissolution systems (A and B) with 6 vessels each were tested. Each vessel was tested with one calibrator tablet in 900 mL deaerated deionized water at 37C with a paddle speed of 50 RPM. Samples were collected promptly at 30 minutes, filtered, and refrigerated until analysis.

In SOFTmax PRO, a Plate section was created wi th Instrument Settings and Data Reduction dialog boxes configured as shown in Figure 1 and Figure 2 (page 3). In the Template Editor, two Groups were created, named Prednisone Stds and Prednisone 50 RPM. The individual standards and samples were assigned to wells in duplicates as illustrated in Figure 3. The standards were assigned a Standards format and the samples were assigned an Unknown format. (The Standards format allows input of the concentration variable and results for Unknowns are automatically calculated by interpolation from the standard curve.)


Pre-read measurements were made with 200 L water in each well of in a Costar UV plate, after which the plate was emptied and blotted dry. Aliquots (varying from 150 L and 275 L) of the standards and samples were transferred into their designated wells using disposable transfer pipets. A cuvette containing deionized water was placed in the cuvette port to serve as the cuvette reference for pathlength correction. The plate was then read in the SPECTRAmax PLUS.

Figure 4 shows the Group Table associated with the associated with the prednisone standards.


Figure 5 shows the plot of mean OD 242 nm vs. concentration. The plot is linear through the highest concentration measured (50 g/mL).


The Group Table associated with the Prednisone 50 RPM samples is shown in Figure 6. Two of the columns (%Dissolved and OUTCOME) were created with custom formulas. The other columns (Values, mean values (renamed Mean OD 242), and results (renamed g/mL) were created automatically by SOFTmax PRO.


Custom formulas and summaries using SOFTmax PRO
To calculate the percent of each tablet that dissolved, a new column named %Dissolved was added to the Group Table for the Plate section, and given the custom formula g/mL*1.8 ( Figure 7 ). For each sp indle, the prednisone concentration (g/mL) of the sample is multiplied by 1.8 (the 1.8 factor was derived from: 900 mL dissolution medium *(1 g/1000 mg) *1/50 mg tablet)*100%.


Two summary lines were added to the bottom of the Group Table, then the USP specifications for Lot L were entered into the summaries. The Calculation dialog boxes for these summary lines are shown in Figure 8. Each formula has been given a short name (sum#1 or sum#2) so that it can be easily referenced in a subsequent formula. The Formula fields contain the minimum (upper part of figure) and maximum (lower part of figure) of the acceptable range for the calibrator Lot L. The Hide Name boxes are checked so that in the final display, only the descriptions and the values from the formulas (in this case, constants) are displayed. The USP specifications in the summary lines can be changed easily for different calibrators, lot numbers or spindle speeds.


The Calculation dialog box for the OUTCOME column, which reports the outcome for each spindle, is shown in Figure 9 . The formula specifies that if the value in the column %Dissolved exceeds the value in Sum#1 (LOT L min spec = 38) and if %Dissolved is less than the value in Sum#2 (LOT L max spec = 48), then the outcome is Pass, otherwise it is Fail.


The OUTCOME column in the Group Table (Figure 6) reveals that all of the spindles passed, except for spindle A03.

The final paragraphs below describe how the overall Summary Section was created. In order to report the results of Apparatus A and Apparatus B separately, they needed to be separated in the Group Table. To accomplish the separation, two new columns were created and the Pass/Fail outcomes were assigned numerical values of 0/10 (Figure 10). The new columns were named HidColA and HidColB (i.e. hidden columns - usually hidden from view to simplify the table).

Note: Columns can be hidden by clicking on the right border of the column header and dragging it to the left border. Hidden columns can be brought into view by selecting Show All from the pop-up menu under the Group heading in SOFTmax PROs toolbar.


The Calculation dialog boxes for the hidden columns are shown in Figure 11. Basically, the formulas use SOFTmax PROs indexing (counting) feature to separate the spindle results for Apparatus A (index numbers 1 through 6) from those of Apparatus B (index numbers 7 through 12). The formulas also use the MakeErr(101) command to return an empty space instead of a value. For example, in HidColA, the formula specifies that if the index is less than or equal to 6 and the OUTCOME for that spindle is Fail, a value of 10 is returned; if the index is 7 or greater, the space is left empty, otherwise a value of 0 is returned. Thus the formula for HIDColA returns values (0 or 10) only for Apparatus A and the formula for HIDColB returns values (0 or 10) only for Apparatus B.


A summary sheet was created in a Notes section, then summary statements derived from applying custom formulas to the data in the Group Table were created. The Calculation dialog boxes for the custom formulas are shown in Figure 12. The logic of the formulas is if any value in the HidCol exceeds 1 (i.e. if a 10 occurs), the overall apparatus fails, otherwise it passes.


Finally, the Apparatus Calibration Summary is shown in Figure 13.

Note: the Notes section has been renamed Summary in this example.


The information contained in the report includes the time and date the plate was read, the overall outcome for each apparatus, type of calibrator tablet and lot number and spindle speed. There is also enough space for the analysts signature. The format of the summary can easily be customized to meet the needs of different laboratories.


CONCLUSION

This application note describes how samples obtained during calibration of a dissolution apparatus can be analyzed quickly and conveniently in a SPECTRAmax PLUS microplate spectrophotometer using the PathCheck feature. Accurate pipetting of the samples into the wells is not necessary; indeed PathCheck corrects for pipetting errors. Using flexible custom formulas and the SOFTmax PROs spreadsheet capability, the data are calculated automatically, and the final results are displayed as overall PASS/FAIL for each apparatus.


REFERENCES
1. The United States Pharmacopoeia, 23rd revision, Second Supplement, 2707 - 2711 (1995).

2. The United States Pharmacopoeia, 23rd revision, 1830-1835 (1995).


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