Total Organic Carbon Analysis

Cleaning Validation: Which TOC Technique is Best?

To ensure quality control and safety in manufacturing facilities within the pharmaceutical industry, it is
important that cleaning validation be conducted following the cleaning of production-related equipment to
ensure the quantity of residual substances collected from the surfaces of the equipment are within
permissible limits. For this cleaning validation using a TOC analyzer, the following 3 types of methods
are available.
(1) Rinse sampling TOC measurement method
(2) Swab sampling aqueous extraction TOC measurement method
(3) Swab sampling direct combustion carbon measurement method
Introduced here are the features of each of these methods, using the TOC-LCPH total organic carbon
analyzer (Shimadzu Corporation, Kyoto, Japan), in the measurement of residual pharmaceutical products
and their constituent substances.
Preparation of Residue Sample
In order to evaluate the cleaning validation sampling methods, residue measurement samples were created
by applying various types of pharmaceutical products and their constituents to stainless steel pots. The
aqueous and non-aqueous substances that were used are listed in Table 1.
Table 1: Sample Types
Substance Name
Tranexamic acid
Anhydrous caffeine
Isopropylantipyrine
Nifedipine
Gentashin ointment
Rinderon ointment

Solubility in Water
Soluble
Soluble
Insoluble
Insoluble
Insoluble
Insoluble

Solvent used Solution Preparation

Water
Water
Ethanol
Acetone
Ethanol
Acetone

The aqueous substances and non-aqueous substances were dissolved in water and ethanol or acetone,
respectively, and the solution concentrations were adjusted to 2000 mgC/L (= carbon concentration of
2000 mg/L). Each residue substance measurement sample consisted of a 5 cm2 area on the surface of a

pot to which a volume of 100 L of each solution was applied and dried. Thus, the amount of carbon in
the sample at each application site was 200 g.
Among these, Gentashin ointment (aminoglycoside antibiotic) and Rinderon ointment (corticosteroid)
were prepared based on determination of their carbon concentrations using the TOC analyzer equipped
with solid sampling module.
(1) Rinse Sampling TOC Measurement Method
The Rinse Sampling TOC Measurement method refers to the method in which the final rinse water used
in the cleaning of a production equipment unit is used as the TOC measurement sample. This method is
suitable for systems that cannot easily be disassembled, such as CIP (clean-in-place) equipment and
narrow tubing. Sampling is considered to be difficult if the residues are not soluble in water.
To evaluate the recovery of the various substances when using the Rinse Sampling TOC Measurement
method, 100 mL of pure water was transferred to the stainless steel pot with its patch of dried sample, and
after stirring for 15 minutes to prepare the rinse solution, TOC measurement was conducted. Some of the
measurement data are shown in Fig. 1.

Figure 1: Measurement Data using Rinse SamplingTOC Measurement Method

Tranexamic Acid
Isopropylantipyrine
Gentashin Ointment
Measurement Conditions
Analyzer:
Shimadzu Total Organic Analyzer TOC-LCPH
Catalyst:
High sensitivity catalyst
Measurement item:
TOC (= TOC by acidification sparge processing)
Calibration curve:
2-point calibration curve using 0 3 mgC/L potassium hydrogen phthalate aqueous
solution
Injection volume:
500 L

Since the carbon content in each of the residue measurement samples is 200 g, the TOC concentration
would be 2 mgC/L if all of the sample were to dissolve in the water.
For the blank, measurement was conducted in the same way using water that was transferred to the
stainless steel pot, which in this case had no patch of dried sample applied to its surface. The measured
blank concentration was subtracted from each TOC concentration, and then compared to the theoretical
value of 2 mgC/L to determine the rate of recovery. The results are shown in Table 2.

Table 2: Measurement Results for Rinse Sampling TOC Measurement Method

Substance Name
TOC Concentration Recovery Rate, [TOC Conc. Blank / Theoretical Conc.]
Blank
0.030
Tranexamic acid
2.14
105%
Anhydrous caffeine
2.19
108%
Isopropylantipyrine
2.20
109%
Nifedipine
2.17
107%
Gentashin ointment
0.117
4.35%
Rinderon ointment
0.333
15.2%

Water-soluble tranexamic acid and water-insoluble anhydrous caffeine had high recovery rates as
expected. Moreover, water-insoluble isopropylantipyrine and nifedipine had high recovery rates.
However, recovery rates of Gentashin ointment and Rinderon ointment were both low, at less than 20%.
From these results, it is clear that evaluation of the rinse water using this method is unreliable due to the
variation of recovery of substances which are not readily soluble in water.
(2) Swab Sampling Water Extraction TOC Measurement Method
The Swab Sampling Water Extraction TOC Measurement method consists of wiping the inside
surface of the production apparatus with a fibrous swab material, extracting the adhering material with
water, and conducting TOC measurement of the extract solution. Since the residue is physically wiped off
from a fixed area of the surface of the apparatus using the swab material, and then analyzed, the sampling
efficiency is high. However, because water is used for extraction of the residue, residues that are
insoluble in water are difficult to extract. Accordingly, cleaning evaluation with respect to these residues
may be difficult for the same reason as that described with respect to difficult-to-dissolve substances in
the (1) Rinse Sampling TOC Measurement method.
To evaluate the recovery of the various substances when using the Swab Sampling Water Extraction
TOC Measurement method, the sample, which was applied to a stainless steel pot, was wiped off with a
5 cm2 piece of fibrous swab material, which was then placed in a glass jar containing 100 mL of pure
water. The residue was then extracted by stirring with a stirrer for 1 hour, after which TOC measurement
was conducted. Some of the measurement data are shown in Fig. 2. Since the fibrous swab material
(Alpha 10 obtained from Texwipe Co.) that was used consists of polyester, very little organic material is
extracted from the swab itself.

Figure 2: Measurement Data using Swab Sampling Water Extraction TOC Measurement Method
Tranexamic Acid
Isopropylantipyrine
Gentashin Ointment
Measurement Conditions
Analyzer:
Shimadzu Total Organic Analyzer TOC-LCPH
Catalyst:
High sensitivity catalyst
Measurement item:
TOC (= TOC by acidification sparge processing)
Calibration curve:
2-point calibration curve using 0 3 mgC/L potassium hydrogen phthalate aqueous
solution
Injection volume:
500 L
Swab material:
5 cm2 piece of Texwipe Alpha 10 swab material washed in pure water and dried

Since the carbon content in each of the residue measurement samples is 200 g, the TOC concentration in
the extraction solution would be 2 mgC/L if all of the sample were wiped off.
For the blank, measurement was conducted in the same way by wiping the stainless pot, which had no
sample applied before conducting extraction. The measured blank concentration was subtracted from each
TOC concentration, and then compared to the theoretical value of 2 mgC/L to determine the rate of
recovery. The results are shown in Table 3.
Table 3: Measurement Results for Swab Sampling Water Extraction TOC Measurement Method
Substance Name
TOC Concentration Recovery Rate, [TOC Conc. Blank / Theoretical Conc.]
Blank
0.059
Tranexamic acid
2.19
107%
Anhydrous caffeine
2.23
109%
Isopropylantipyrine
1.90
92.2%
Nifedipine
1.86
89.9%
Gentashin ointment
0.093
1.70%
Rinderon ointment
0.208
7.45%

Water-soluble tranexamic acid and water-insoluble anhydrous caffeine had high recovery rates as
expected. Moreover, water-insoluble isopropylantipyrine and nifedipine had high recovery rates of about
90%. However, recovery rates of Gentashin ointment and Rinderon ointment were both low, at less than
10%. From these results, it is clear that evaluation of the rinse water using this method is unreliable due to
the variation of recovery of substances which are not readily soluble in water.
(3) Swab Sampling Direct Combustion Method
The Swab Sampling Direct Combustion method consists of wiping the inside surface of the production
apparatus with a piece of inorganic quartz filter paper swab material, and then conducting measurement
using a direct combustion carbon measurement system. The swab material with adhering residue is
merely placed in the sample boat, and the carbon content is measured directly by the TOC analyzer with a
connected Solid Sample Combustion Unit (SSM-5000A, Shimadzu Corporation, Kyoto, Japan). Using
this method, water-insoluble residues that are difficult to extract in water can also be collected, and
measurement can be quickly and easily conducted without the need for any pretreatment, such as sample
extraction, etc.

To evaluate the rate of recovery of the different types of substances using the Swab Sampling Direct
Combustion method, we used the quartz glass filter paper swab material to wipe the sample off the
sample adhering to the stainless steel pot, placed the swab in the SSM-5000A sample boat, and conducted
TC measurement. Some of the measurement data are shown in Fig. 5.

Figure 3: Measurement Data using Swab Sampling Direct Combustion Method

Tranexamic Acid
Isopropylantipyrine
Gentashin Ointment
Measurement Conditions
Analyzer:
Shimadzu Total Organic Analyzer TOC-LCPH + SSM-5000A Solid Sample
Combustion Unit (IC circuit bypass using system with cell switching valve set)
Cell length:
Short cell
SSM carrier gas:
400 mL/min oxygen gas
Measurement item:
TC
Calibration curve:
1-point calibration curve using 1% C glucose aqueous solution
Swab material:
Advantec quartz glass paper QR-100 (diameter 45 mm) heat-treated at 600 C for 15
minutes

Since the carbon content in each of the residue measurement samples is 200 g, the TC value would be
200 g if all of the sample were wiped off. For the blank, measurement was conducted in the same way
by wiping the stainless pot, which had no sample applied. The measured blank value was subtracted from
each TC value, and then compared to the theoretical value of 200 g to determine the rate of recovery.
The results are shown in Table 4. A high recovery rate of about 100% was obtained for all the substances,
regardless of whether they were water-soluble or water-insoluble.
Table 4: Measurement Results for Swab Sampling Direct Combustion Method
Substance Name
TOC Value [C]
Recovery Rate, [TC Value Blank / Theoretical Value]
Blank
0.00
Tranexamic acid
202
101%
Anhydrous caffeine
201
100%
Isopropylantipyrine
210
105%
Nifedipine
212
106%
Gentashin ointment
200
100%
Rinderon ointment
209
104%

Conclusion
The measurement methods used here and their respective recovery rates are summarized in Table 5.
When using the Rinse Sampling TOC Measurement and the Swab Sampling Water Extraction TOC
Measurement methods, substances that do not easily dissolve in water were found to include those that
had both high and low recovery rates. It is thought that this may be due to differences in the strength with
which the substances adhere to the stainless steel pot. Accordingly, it is probable that residue evaluation
using these methods would be difficult for substances with low recovery rates.
In contrast, high recovery rates were obtained for all the substances when using the Swab Sampling
Direct Combustion method, regardless of whether the substances were water-soluble or water-insoluble,
thereby permitting residue evaluation. Therefore, this method is considered to be the most effective
measurement method for conducting cleaning validation, especially when multiple compounds are being
manufactured in the same vat.
Table 5: Summary of Measurement Results

Substance Name
Tranexamic acid
Anhydrous caffeine
Isopropylantipyrine
Nifedipine
Gentashin ointment
Rinderon ointment

Solubility in
Water
Soluble
Soluble
Insoluble
Insoluble
Insoluble
Insoluble

Shimadzu Scientific Instruments

7102 Riverwood Drive, Columbia, MD 21046
Phone: 800-477-1227, Fax: 410-381-1222
www.ssi.shimadzu.com
[email protected]

Rinse Sampling
TOC Measurement
Method
105%
108%
109%
107%
4.35%
15.2%

Recovery Rate
Swab Sampling Water
Extraction TOC
Measurement Method
107%
109%
92.2%
89.9%
1.70%
7.45%

Swab Sampling
Direct Combustion
Method
101%
100%
105%
106%
100%
104%