A novel approach to cleaning validation for

pharmaceutical manufacturing by online supercritical

fluid extraction/supercritical fluid chromatography

1. 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.

3. 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.
(l) UCX-RP

(lll) UCX-SIL

mAU

mAU

RT : 7.87 min
N : 16454
Tf : 1.767

60
50

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

50
1.0

2.0

3.0

4.0

mAU
60

mAU
130
120
110
100
90
80
70
60
50
40
30
20
10
0
-10
-20

RT : ND
N : ND
Tf : ND

40
35
30
25
20
15

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).

5-30%B (0-7 min)

(lV) UCX-DIOL

45

Materials and Reagents

5-40%B (0-7 min)

0.0

12.0 min

10
5
0
-5
-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

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0 min

RT : 7.96 min
N : 48053
Tf : 0.692

0.0

12.0 min

Time program:
5-50%B (0-7 min)

(ll) UCX-GIS
55
50

2. Experimental

RT : 8.56 min
N : 59056
Tf : 0.693

130
120
110
100
90
80
70
60
50
40
30
20
10
0
-10

40

Injected sample:
1 L of 1% Standard sample

B Conc. [%]

Kenichiro Tanaka1, William Hedgepeth1, Daisuke Nakayama2, Hidetoshi Terada2,

Minori Nakashima2, Tadayuki Yamaguchi2
1: Shimadzu Scientific Instruments, Inc., MD, USA
2: Shimadzu Corporation, Kyoto, Japan

1.0

2.0

3.0

4.0

40
30
20
10
0
0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

5
10
Time [min]

15

Abbreviations:
RT : Retention time
N : Theoretical plate
Tf : Tailing factor (5%)

12.0 min

Figure 2. Comparison of columns for SFC analysis of standard detergent sample

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.

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.
(l) Linearity test
mAU
110
100
90

1A: Supercritical fluid chromatography (SFC) system for analytical method development

80
70
60

CO2 delivery unit

50

Peak Area (x100,000)

Sample concentration:
1%
0.50%
0.10%
0.05%
0.01%
0.005%
0%

12.0
10.0
8.0
6.0

40

Autosampler

Column oven
with column selector

Photodiode
array detector

Injected sample:
2 L of 0 to 1% Standard samples

30
20

Time program:
10%B (0-2 min), 10-60%B (2-7 min),
10%B (9-13 min)

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. (%)

B Conc. [%]

0.0

(ll) Reproducibility test

Back pressure
regulator

Solvent
delivery unit

mAU
60

Reproducibilities
(n=6, %RSD)

50
40

Sample

30
20

CO2

Modifier

10

Retention
time

Area

0.17

2.46

0.10% standard
sample

60
50
40
30
20
10
0
0

6 8 10 12 14
Time [min]

-10

1B: On-line Supercritical fluid extraction/chromatography (SFE/SFC) system

-20
0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0 min

CO2 delivery unit

SFE unit

Column oven

Figure 3. Performance evaluations of SFC analytical method

Photodiode
array detector

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).
Back pressure
regulator

Solvent delivery
unit with
modifier selector
CO2

(l) Sequential diagram of on-line SFE/SFC

Extraction vessel
containing sample swab

Static
Extraction
(3 min)

Modifiers

Dynamic
Extraction
(3 min)

SFC
Analysis
(13 min)

60
50

B Conc. [%]

1C: Static and Dynamic extraction

SFE unit
Extraction
vessel

40
30
20
10

From
Pumps

To
Colum

1: Standby

-6
2: Static extraction

3: Dynamic extraction

4: Analysis

Figure 1. Flow diagrams of Nexera UC system

3
Time [min]

Static
Extraction

Dynamic
Extraction

SFC
Analysis

Spiked sample amount:

Alkylbenzenesulfonate

2.0

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 m)

(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
: 40C
: 15 MPa
: 220 nm
: Shown in figure

1.50

200 g

1.25

100 g

1.00

20 g

[Sample Preparation]
A total of 10 to 500 g 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 m)
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. : 40C
Back pressure : 15 MPa
Wavelength
: 220 nm

0.75
0.50

10 g

0.5

R2 = 0.996

0.25
0.00

0
-5.0

-2.5

0.0

2.5

5.0

7.5

100

200

300

400

500 g/Swab

min

(lll) Reproducibility test

mAU
2.0

Static
Extraction

Dynamic
Extraction

SFC
Analysis

1.5

Table 2 On-line SFE/SFC Analytical conditions

12

Peak Area(x10,000,000)

500 g

50 g

1.5

1.0

Column

mAU

2.5

Table 1 SFC Analytical conditions

(ll) Linearity test

3.0

Analytical conditions

-3

Sample
1.0

100 g standard
sample

0.5

Reproducibilities
(n=5, %RSD)
Retention
time

Area

0.19

5.76

-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

4. 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.

5. Acknowledgement
The sample was provided by DAIICHI SANKYO COMPANY, LIMITED.