T.  Kanashova,  D.  Wilhelm,  Copyright  AnaTox,  Germany,  2011     1  

Application Note

Validation  of  Online  Dissolution  with  the  ADS  100  and  the  Agilent  1260  HPLC-­‐System  

 Tamara  Kanashova,  Detlef  Wilhelm*,  AnaTox  GmbH  &  Co.  KG,  Fuerstenwalde,  Germany  

Introduction Prednisone is commonly used in laboratories as a dissolution bath calibrator. Manual dissolution is a very time intensive process; the automation of this technique is highly desirable. In this study, the ADS 100 is used as a tool to automate the Analytical Instrumental Qualification of Dissolution Bath according USP <1058> specifications and initiating the analysis with HPLC. In addition to reduction of the hands-on time involved in dissolution, ADS Control software provides the documentation of the automated dissolution process. The objective of this study is to determine the equivalency between manual and automated dissolution sampling results, the precision and linearity of the HPLC-injector working as online sampling collector and injector with the ADS 100 and the correct assignment of the samples in the rack/wellplate (1).

Material and Methods The study was run using USP Prednisone tablets Lot P1I300 containing 10 mg prednisone. Distilled degassed water (HPLC grade) was used for preparing the dissolution media according to the USP specifications. The samples taken by the ADS 100 were filtered under

pressure. The samples were taken 0, 5, 10, 15, 20 and 25 minutes after the start of the dissolution experiment for the determination of correct assignment of the samples in the rack. Both, the manual and automated samples were taken 30 minutes after start of the dissolution experiment to determine the dissolution of prednisone according to the USP criteria. All samples were analyzed by

HPLC according to the procedure contained in the USP manual method on the same UV-spectrophotometer (HPLC-DAD). The devices used in this application note are listed in Table 1.

 

 

*  Author  to  should  be  corresponded:  info@anatox.de  

 

T.  Kanashova,  D.  Wilhelm,  Copyright  AnaTox,  Germany,  2011     2  

Table 1: Devices

Device Description

Dissolution-Bath SR8 PLUS Dissolution Test Station, Hanson Research, USA Dissolution-Sampler ADS 100, AnaTox, Germany

Degasser 1260 µ-Degasser, Agilent Technologies, Germany

Pump 1260 Binary Pump, Agilent Technologies, Germany

Injector 1260 WellPlate-ALS, Agilent Technologies, Germany

Colum oven 1290 Infinity TCC, Agilent Technologies, germany

Detector 1260 DAD, Agilent Technologies

Vacuum pump Alcatel, Germany

Restriction capillary 2m * 0,12mm stainless steel capillary

The samples were analyzed using a restriction capillary (see Table 1) at 40°C in a thermostated column compartment. The flow rate of the binary HPLC pump was set isocratic (100% water) to 1ml/min, the detection wavelength of the DAD to 242nm (absorption maximum of prednisone). The injection of the samples (50µl) was carried out by using the same HPLC automatic sampler already used for sample collecting (wellplate sampler, G1367C). The ADS 100 configuration consists of 6 circulating pumps, one flushing pump, a 10-position valve and a flow sensor. The ADS 100 settings for transferring samples are described in Table 2. The samples taken by the ADS (250µl) were collected in a 96-position wellplate and analyzed by HPLC after collection of all samples (asynchronous). The manually taken samples were taken with glass pipettes and filled into HPLC vials.

Table 2: ADS 100 Settings

Setting Description

Flow rate 5 ml/min Sampling volume 250 µl Analysis asynchron

Results At the 30 minute sampling event, the mean results of the samples taken with ADS 100 and of the manually taken samples for 6 vessels are essentially identical. The dissolved prednisone concentration was in the USP range (see Figure 1).

Figure 1: Dissolution rates depending on manual or automatic taken samples

 

T.  Kanashova,  D.  Wilhelm,  Copyright  AnaTox,  Germany,  2011     3  

The correct assignment of the samples in the rack can be monitored by the increasing concentration of prednisone with the experiment time (see Figure 2). With the Analytical Instrument Qualification procedure, the absence of concentration spreading during sampling with the dissolution sampler was proven.

Figure 2: Proof of correct assignment of the samples by increased concentration with time and sampling position

The influence of exchanging valuable parts (e.g. injection valve) of the Auto injector was evaluated by checking the precision and linearity of standard injections. Figure 3 shows no decrease in precision after exchanging parts from the standard configuration of the wellplate sampler to the assignment as a collector and injection device. No influence on the linearity was detected by injecting increasing concentrations of prednisone solutions.

Figure 3: No influence on injection precision after exchange of valuable parts of the Auto injector

Figure 4 shows the correlation curve with the data for regression showing no influence of the changed configuration.

Figure 4: No influence on linearity after exchange of valuable parts of the Auto injector Conclusion The validation of online dissolution with the ADS100 in conjunction with a wellplate sampler of the Agilent 1260 HPLC series was performed. The results after analysis by HPLC using the prednisone calibrator tablets and prednisone solutions show the ability of

 

 

 

T.  Kanashova,  D.  Wilhelm,  Copyright  AnaTox,  Germany,  2011     4  

automation of dissolution testing with HPLC. No influence of the modification of the wellplate sampler for dissolution purposes was detected. All data (e.g. injection precision and linearity) show the same specification as for the standard configuration. This proofs the usage of the ADS 100 in conjunction with the wellplate sampler of Agilent Technologies for automation of dissolution experiments. The AIQ tool of AnaTox can be used to qualify the whole system used for automated dissolution. Each chromatography software can be used that is able to control the “rest” of the HPLC system and to detect the compounds used for dissolution experiments. References:

(1) Bansal, S.K., Layloff, T., Bush, E.D., Hamilton, M., Hankinson, E.A., Landy, J.S., Lowes, S., Nasr, M.M., Jean, P.A., Shah, V.P. (2004). Qualification of Analytical Instruments for Use in the Pharmaceutical Industry: A Scientific Approach. AAPS PharmSciTech. 22, p. 1-8

(2) Kromidas, S. (2000). Handbuch Validierung in der Analytik. Wiley-VCH Verlag GmbH, Weinheim. P. 46-364