PO-CON1575E
On-line supercritical fluid extraction/ supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
HPLC 2015
PSA-SFC-14
Daisuke Nakayama, Hidetoshi Terada, Minori Nakashima, Tadayuki Yamaguchi Shimadzu Corporation, Kyoto, Japan
On-line supercritical fluid extraction/supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
Introduction The cleaning validation is necessary to establish the quality and safety of pharmaceutical drug products. In the cleaning validation protocols, the direct sampling is performed with swabs, which are sticks with textiles at one side. The sample on the swab after the swabbing on the surface of equipment is analyzed with TOC analyzer and HPLC. Recently, HPLC has been more preferable because of the growing need for the individual analysis of
products. Before the HPLC analysis, manual processes such as a sample extraction and a sample condensation are required. Such manual processes may affect to the quality of results. Thus, we evaluated the application of a novel on-line supercritical fluid extraction/chromatography system for the cleaning validation.
Experimental Materials and Reagents A commercially available detergent containing alkylbenzene sulfonates was diluted with methanol and used as a standard sample. For the test of sample extraction, the sample was dropped onto a swab (ITW Texwipe, USA).
System Nexera UC system (Shimadzu corporation, Japan) was used for both the screening of the method using supercritical fluid chromatography (SFC) (Figure 1A) and the supercritical fluid sample extraction (SFE) followed by SFC directly (SFE/SFC) (Figure 1B). The schematic diagram for static and dynamic extraction of supercritical fluid extraction unit was shown in Figure 1C.
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On-line supercritical fluid extraction/supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
A: Supercritical fluid chromatography (SFC) system for analytical method development CO2 delivery unit Autosampler
Column oven with column selector
Photodiode array ditector
Back pressure regulator
Solvent delivery unit
CO2
Modifier
B: On-line Supercritical fluid extraction/chromatography (SFE/SFC) system CO2 delivery unit SFE unit
Photodiode array ditector
Back pressure regulator
Solvent delivery unit with modifier selector CO2
Column oven
Extraction vessel enclosing sample swab
Modifiers
C: Static and Dynamic extraction SFE unit Extraction vessel
From Pumps
To Colum
1: Standby
2: Static extraction
3: Dynamic extraction
4: Analysis
Figure 1. Flow diagrams of Nexera UC systems
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On-line supercritical fluid extraction/supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
Analytical conditions Table 1 SFC Analytical conditions Column Mobile Phase Time program Flow Rate Column Temp. Back pressure Wavelength Injection Vol.
: Shim-pack UCX series columns (250 mm L. x 4.6 mm I.D., 5 μm) (i) UCX-RP (ODS with polar group), (ii) UCX-GIS (ODS), (iii) UCX-SIL, (iv) UCX-DIOL : A: CO2; B: Methanol : Shown in the figure : 3.0 mL/min : 40 °C : 15 MPa : 220 nm : Shown in figure
Table 2 On-line SFE/SFC Analytical conditions Sample Preparatation A total of 10 to 500 µg standard samples in methanol were dropped onto a total of three swabs. The swabs were enclosed into an extraction vessel and set to the SFC unit. Static extraction Extraction time Mobile phase B conc. Flow rate Back pressure
: 3 min : A: CO2; B: 0.1% (w/v) ammonium formate in methanol : 10% : 3.0 mL/min : 15 MPa
Dynamic extraction Extraction time Mobile phase B Conc. Flow rate Back pressure
: 3 min : A: CO2; B: Methanol : 10% : 3.0 mL/min : 15 MPa
SFC Column Mobile Phase Time program Flow Rate Column Temp. Back pressure Wavelength
: Shim-pack UCX-SIL (250 mm L. x 4.6 mm I.D., 5 μm) : A: CO2; B: Methanol : 10%B (0-2 min), 10-60%B (2-7 min), 60%B (7-9 min), 10%B (9-13 min) : 3.0 mL/min : 40 °C : 15 MPa : 220 nm
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On-line supercritical fluid extraction/supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
Results Analytical method development The analytical method for SFC was developed by screening four columns and gradient conditions. The UCX-SIL column showed the excellent peak shape (Figure 2) and was used for further analysis. (i) UCX-RP
(iii) UCX-SIL
mAU
mAU
50 40 30 20 10 0 -10 0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
1.0
2.0
3.0
5-50%B (0-7 min) 5-30%B (0-7 min) 50
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0 min
(iv) UCX-DIOL mAU 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20
RT : ND N : ND Tf : ND
0.0
Time program: 5-40%B (0-7 min)
0.0
12.0 min
(ii) UCX-GIS mAU 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 -10
RT : 8.56 min N : 59056 Tf : 0.693
130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
RT : 7.96 min N : 48053 Tf : 0.692
0.0
12.0 min
1.0
2.0
3.0
4.0
B Conc. [%]
RT : 7.87 min N : 16454 Tf : 1.767
60
Injected sample: 1 µL of 1% Standard sample
40 30 20 10 0 0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0 min
5 10 Time [min]
15
Abbreviations: RT : Retention time N : Theoretical plate Tf : Tailing factor (5%)
Figure 2. Comparizon of columns for SFC analysis of standard detergent sample
The time program was developed in consideration of on-line SFE/SFC (data not shown). The optimized time program and the result of performance evaluations were shown in Figure 3. (i) Lineartity test mAU 110 100 90 80 70 60 50
Peak Area (x100,000)
Sample concentration: 1% 0.50% 0.10% 0.05% 0.01% 0.005% 0%
12.0
Injected sample: 2 µL of 0 to 1% Standard samples
10.0 8.0 6.0
40
Time program: 10%B (0-2 min), 10-60%B (2-7 min), 10%B (7-13 min)
30 20
4.0
R2 = 0.998
10 0
2.0
-10 1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
min
0 0.00
0.25
0.50
0.75
1.00 Conc. (%)
(ii) Reproducibility test mAU 60
Sample
50 40 30
Reproducibilities (n=6, %RSD) Retention time
Area
0.17
2.46
0.10% standard sample
0
60 50 40 30 20 10 0 0
20 10
B Conc. [%]
0.0
2
4
6 8 10 12 14 Time [min]
-10 -20 0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0 min
Figure 3. Performance evaluations of SFC analytical method 5
On-line supercritical fluid extraction/supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
On-line SFE/SFC analysis of detergent in swab Based on the optimized SFC analytical condition, an on-line SFE/SFC analytical condition was developed and its performance was evaluated (Figure 4). (i) Sequencial diagram of on-line SFE/SFC Static Extraction (3 min)
Dynamic Extraction (3 min)
SFC Analysis (13 min)
60 B Conc. [%]
50 40 30 20 10 0 -6
-3
0
3 Time [min]
6
9
12
(ii) Linearity test mAU 3.0
Static Extraction
Dynamic Extraction
SFC Analysis
Spiked sample amount:
Alkylbenzenesulfonate
2.5
2.0
1.5
500 µg
1.50
200 µg
1.25
100 µg
1.00
50 µg 20 µg
1.0
10 µg
0.5
Peak Area (x10,000,000)
0.75 0.50
0.00
0 -5.0
-2.5
0.0
2.5
5.0
7.5
R2 = 0.996
0.25 0
100
200
300
400
500 µg/Swab
min
(iii) Reproducibility test mAU 2.0
Static Extraction
Dynamic Extraction
SFC Analysis
1.5
Sample 1.0
100 µg standard sample
0.5
Reproducibilities (n=5, %RSD) Retention time
Area
0.19
5.76
0 -5.5
-2.5
0.0
2.5
5.0
7.5
min
Figure 4. On-line SFE/SFC analysis of standard detergent sample-spiked swabs
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On-line supercritical fluid extraction/supercritical fluid chromatography: A novel approach to the cleaning validation for pharmaceutical manufacturing
Conclusion The evaluation results showed that the on-line SFE/SFC can provide the reliable data for the cleaning validation for pharmaceutical manufacturing. The benefit of on-line SFE/SFC analysis is not only the data reliability but also convenience and safety by eliminating manual extraction process. It may streamline laboratory processes and improve productivity and safety of pharmaceutical manufacturing facilities.
Acknowledgement The sample was provided by DAIICHI SANKYO COMPANY, LIMITED.
First Edition: June, 2015
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