This document describes a novel approach using online supercritical fluid extraction/chromatography (SFE/SFC) for cleaning validation in pharmaceutical manufacturing. The system allows direct extraction and analysis of residue samples on swabs without manual sample preparation. Analytical methods were developed using different columns and conditions. Testing showed the SFE/SFC system provided reliable quantitative analysis of detergent residues down to 10 μg with good linearity and reproducibility. The online system eliminates manual extraction steps, improving productivity, reliability and safety for cleaning validation.

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A Novel Approach to Cleaning Validation for Pharmaceutical
Manufacturing:
Using Online Supercritical Fluid Extraction/Supercritical Fluid
Chromatography
Kenichiro Tanaka1, William Hedgepeth1, Daisuke Nakayama2, Hidetoshi Terada2, Minori
Nakashima2, Tadayuki Yamaguchi2
1: Shimadzu Scientific Instruments, Inc., MD, USA, 2: Shimadzu Corporation, Kyoto, Japan

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Introduction
Cleaning validation is necessary to establish the quality and safety of pharmaceutical drug
products.
In cleaning validation protocols, direct sampling is performed with swabs, which are sticks with
textiles at one end.
The sample on the swab after swabbing the surface of equipment is analyzed with a 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.

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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
is shown in Figure 1C.

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Figure 1: Flow diagrams of
Nexera UC system
System

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Analytical Conditions
Column : 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
Mobile Phase : A: CO2; B: Methanol
Time program : Shown in the figure
Flow Rate : 3.0 mL/min
Column Temp. : 40°C
Back pressure : 15 MPa
Wavelength : 220 nm
Injection Vol. : Shown in figure
Table 1: SFC Analytical conditions

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[Sample Preparation]
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 SFE/SFC unit.
[Static extraction]
Extraction time : 3 min
Mobile phase : A: CO2; B: 0.1% (w/v) ammonium formate in methanol
B conc. : 10%
Flow rate : 3.0 mL/min
Back pressure : 15 MPa
[Dynamic extraction]
Extraction time : 3 min
Mobile phase : A: CO2; B: Methanol
B Conc. : 10%
Flow rate : 3.0 mL/min
Back pressure : 15 MPa
[SFC]
Column : Shim-pack UCX-SIL (250 mm L. x 4.6 mm I.D., 5 µm)
Mobile Phase : A: CO2; B: Methanol
Time program : 10%B (0-2 min), 10-60%B (2-7 min), 60%B (7-9 min), 10%B (9-13 min)
Flow Rate : 3.0 mL/min
Column Temp. : 40°C
Back pressure : 15 MPa
Wavelength : 220 nm
Table 2: On-line SFE/SFC Analytical conditions
Analytical Conditions

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Results
Analytical method development
The analytical method for SFC was developed by screening four columns and gradient conditions. The
UCX-SIL column showed excellent peak shape (Figure 2) and was used for further analysis.
Figure 2: Comparison of columns for SFC analysis of standard detergent sample
0.0 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
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
mAU
0.0 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
-10
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
mAU
0.0 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
-10
0
10
20
30
40
50
60
mAU
0.0 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
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
mAU
(l) UCX-RP
0
10
20
30
40
50
0 5 10 15
BConc.[%]
Time [min]
(lll) UCX-SIL
(ll) UCX-GIS (lV) UCX-DIOL
5-50%B (0-7 min)
Time program:
5-40%B (0-7 min)
5-30%B (0-7 min)
RT : 7.87 min
N : 16454
Tf : 1.767
RT : ND
N : ND
Tf : ND
RT : 8.56 min
N : 59056
Tf : 0.693
RT : 7.96 min
N : 48053
Tf : 0.692
Abbreviations:
RT : Retention time
N : Theoretical plate
Tf : Tailing factor (5%)
Injected sample:
1 µL of 1% Standard sample

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Results
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.
Figure 3: Performance evaluations of SFC analytical method
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
-20
-10
0
10
20
30
40
50
60
mAU
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
-10
0
10
20
30
40
50
60
70
80
90
100
110
mAU
(l) Linearity test
(ll) Reproducibility test
1%
Sample concentration:
0.50%
0.10%
0.05%
0.01%
0.005%
0%
0.00 0.25 0.50 0.75 1.00
0
2.0
4.0
6.0
8.0
10.0
12.0
Peak Area (x100,000)
Conc. (%)
R2 = 0.998
Sample
Reproducibilities
(n=6, %RSD)
Retention
time
Area
0.10%
standard
sample
0.17
2.46
Time program:
10%B (0-2 min), 10-60%B (2-7
min), 10%B (9-13 min)
Injected sample:
2 µL of 0 to 1% Standard
samples
0
20
40
60
0 2 4 6 8 101214
BConc.[%]
Time [min]

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Results
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).
Figure 4: On-line SFE/SFC analysis of standard detergent sample-spiked swabs
(l) Sequential diagram of on-line SFE/SFC
0
10
20
30
40
50
60
-6 -3 0 3 6 9 12
BConc.[%]
Time [min]
SFC
Analysis
(13 min)
Dynamic
Extraction
(3 min)
Static
Extraction
(3 min)

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Results
Figure 4: On-line SFE/SFC analysis of standard detergent sample-spiked swabs
50 µg
100 µg
200 µg
500 µg
Spiked sample amount:
(ll) Linearity test
0
100
200
300
400
µg/Swab
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Peak Area
(x10,000,000)
500
R2 = 0.996
SFC
Analysis
Dynamic
Extraction
Static
Extraction
0
0.5
1.0
1.5
2.0
2.5
3.0
mAU
-5.0 -2.5 0.0 2.5 5.0 7.5 min
Alkylbenzenesulfonate
10 µg
20 µg
(lll) Reproducibility test
SFC
Analysis
Dynamic
Extraction
Static
Extraction
0
0.5
1.0
1.5
2.0
mAU
-5.5 -2.5 0.0 2.5 5.0 7.5 min
Sample
Reproducibilities
(n=5, %RSD)
Retention
time
Area
100 µg
standard
sample
0.19
5.76

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Conclusion
The evaluation results showed that the on-line SFE/SFC can provide reliable data for the
cleaning validation for pharmaceutical manufacturing.
The benefit of on-line SFE/SFC analysis is not only data reliability but also the convenience
and safety gained by eliminating the manual extraction process.
It may streamline laboratory processes and improve productivity and safety of
pharmaceutical manufacturing facilities.
The sample was provided by DAIICHI SANKYO COMPANY, LIMITED.
Acknowledgement

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