Application Note 12.99.
2 December 1999
World Leader
in
Total Organic Carbon
Analysis
Model A-2000 Wide Range Total Organic Carbon Analyzer
Total Organic Carbon Analysis for Cleaning Validation
in Pharmaceutical Manufacturing
INTRODUCTION
In the pharmaceutical industry, Good Manufacturing
Practices (GMP) require that the cleaning of drug
manufacturing equipment be validated.1 Many
different validation techniques can demonstrate that
the manufacturing equipment is cleaned and
essentially free from residual active drug substances
and all cleaning agents.
Common analytical techniques in the validation
process include HPLC, spectrophotometry (UV/Vis)
and TOC. HPLC and UV/Vis are classified as
specific methods that identify and measure
appropriate active and substances. TOC is classified
as a non-specific method and is ideal for detecting
all carbon-containing compounds, including active
species, excipients, and cleaning agent(s).
The disadvantage of specific methods, particularly
HLPC, is that a new procedure must be developed for
every active drug substance that is manufactured.
This development process can be very time
consuming and tedious, plus important sampling
issues also must be considered. In addition, HPLC
analyses must be performed in a relatively short time
period after sampling to avoid any chemical
deterioration of the active substance. Finally, the
sensitivity of HPLC methods can be limited by the
presence of degradation products.
TOC analysis can be adapted to any drug compound
or cleaning agent that contains carbon. The method
is sensitive to the ppb range and is less time
consuming than HPLC or UV/Vis. USP TOC
methods are standard for Water for Injection and
Purified Water2, and simple modifications of these
methods can be used for cleaning validation.
Anatel Corporation
2200 Central Avenue, Boulder, CO 80301; Phone (303) 442-5533 Fax (303) 447-8365; E-mail: info@anatel.com; http://www.anatel.com
©Copyright 1999, Anatel Corporation. All Rights Reserved. Printed in USA.
Information, descriptions and specifications are subject to change without notice.
METHODOLOGY
TOC analysis involves the oxidation of carbon and
the detection of the resulting carbon dioxide. A
number of different oxidation techniques exist,
including photocatalytic oxidation, chemical
oxidation, and high-temperature combustion. In this
study, an Anatel A-2000 Wide-Range TOC Analyzer,
equipped with an autosampler, was used. The Anatel
A-2000 Wide-Range Analyzer measures TOC in
accordance with ASTM methods D 4779-88 and
D 4839-88. It measures TOC directly by adding
phosphoric acid to the water sample to reduce the pH
to approximately 2 to 3. At this low pH any inorganic
carbon that is present is liberated as CO2 into a
nitrogen carrier gas and is directly measured by a
non-dispersive infrared (NDIR) detector. Any
remaining carbon in the sample is assumed to be
TOC. A sodium persulfate oxidant is then added to
the sample, and in the presence of UV radiation, the
remaining carbon is oxidized to CO2. The amount of
CO2 generated is then measured by the NDIR to
determine the amount of TOC originally present in
the water.
Key Words:
Cleaning Validation
Total Organic Carbon
Method Detection Limit
Precision and Accuracy
Linearity
Swab Recovery
Application Note 12.99.2 December 1999

For equipment cleaning validation there are two types

10000
of TOC sampling techniques. One is the direct 9000 y = 19.132x + 51.042

Measured TOC (ppb)

2
surface sampling of the equipment using a swab. The 8000 R = 0.9998
7000
second consists of a final rinse of the equipment with 6000
high-purity water (typically <500 ppb TOC) and 5000
4000
collecting a sample of the rinse for analysis. In 3000
general, direct surface sampling indicates how clean 2000
1000
the actual surface is. The purpose of this study is to 0
demonstrate the performance of the Anatel A-2000 0 100 200 300 400 500

TOC analyzer to measure a variety of different CIP 200 Concentration (ppm)

organic residues on stainless steel surfaces.

Performance parameters tested include linearity, Figure 2: Linearity of CIP-200
method detection limit (MDL), limit of quantitation
(LOQ), accuracy, precision, and swab recovery. 45

40 y = 0.0355x + 1.1983
2
LINEARITY R = 0.9787

Measured TOC (ppm)

35

30

TOC analysis should provide a linear relationship 25

20
between the measured compound concentration and 15
the TOC response of the analyzer. We evaluated four 10

different types of cleaning agents for linearity: 5

CIP-100 (alkaline), CIP-200 (acidic), Alconox 0

0 200 400 600 800 1000
(emulsifier) and Triton-X 100 (wetting agent). Alconox Concentration (ppm)

Results are shown in Figures 1-4. Correlation

coefficients ranged from 0.9787 to 0.9998. Alconox Figure 3: Linearity of Alconox
and Triton-X 100 have a tendency to “foam”
depending on the concentrations that are analyzed
and this “foaming” phenomena can have a negative
12500
effect on the accuracy of the TOC result (reduced
Measured TOC (ppb)

y = 415.76x + 16.997
R2). Three representative examples of active 10000 2
R = 0.9982
substances were also tested for linearity: an excipient 7500
(sucrose), an antibiotic (vancomycin), and endotoxin. 5000
Results are shown in Figures 5-7. All three
2500
compounds demonstrated excellent linearity with
0
correlation coefficients (R2) ranging from 0.9996 to
0 5 10 15 20 25
0.9998.
Triton-X 100 Concentration (ppm)

10000 Figure 4: Linearity of Triton-X 100

9000 y = 39.254x + 1.462
8000 2
R = 0.9997
Measured TOC (ppb)

7000
6000
5000
4000 12000
Measured TOC (ppb)

10000 y = 1.003x + 45.185

3000
8000 R2 = 0.9996
2000
1000 6000
0 4000
0 50 100 150 200 250 2000
CIP 100 Concentration (ppm)
0
0 2000 4000 6000 8000 10000 12000
Sucrose Concentration (ppb)
Figure 1: Linearity of CIP-100

Figure 5: Linearity of sucrose

Anatel Corporation
2200 Central Avenue, Boulder, CO 80301; Phone (303) 442-5533 Fax (303) 447-8365; E-mail: info@anatel.com; http://www.anatel.com
©Copyright 1999, Anatel Corporation. All Rights Reserved. Printed in USA.
Information, descriptions and specifications are subject to change without notice.
Application Note 12.99.2 December 1999

8000

Measured TOC (ppb)

y = 0.8758x + 62.133 Vial Number Average TOC (ppb)
2
6000 R = 0.9998
1 58
4000 2 72
3 75
2000
4 93
0 5 79
0 2000 4000 6000 8000 6 102
Vancomycin Concentration (ppb) 7 60
8 83
Figure 6: Linearity of vancomycin 9 67
10 54
Average 74.3
Standard Deviation 15.5
8000 MDL (Student t, n=10) 50 ppb
7000
Measured TOC (ppb)

y = 0.9287x + 30.8
LOQ 151 ppb
6000 R2 = 0.9998
5000 Table 1: Calculated TOC averages from 10 blank
4000
3000
vials
2000
1000
0
PRECISION AND ACCURACY
0 2000 4000 6000 8000
To demonstrate the precision and accuracy for this
Endotoxin Concentration (ppb)
TOC method, a representative solution of CIP-100 as
1000 ppb, or 1 ppm as carbon, was analyzed
Figure 7: Linearity of endotoxin sequentially ten times. This carbon concentration was
chosen to evaluate these method parameters because,
in general, TOC residual limits are typically around
METHOD DETECTION LIMIT AND 1 ppm. Results are listed in Table 2. At this TOC
LIMIT OF QUANTITATION level, the precision was ± 1% and the accuracy was
± 5%.
We determined the method detection limit (MDL) by
measuring the TOC response of the method blank. Vial Number Measured TOC (ppb)
A method blank consists of the sampling vial, swab, 1 1041
and recovery solution. In this study the recovery
1 1025
solution was low TOC (< 25 ppb) water. 10 pre-
1 1039
cleaned vials were filled with of the low TOC water.
One swab was placed in each vial (Texwipe Alpha 1 1057
Swab TX761; tips cut off). Solutions were vortexed 1 1054
and allowed to stand for one hour prior to analysis. 2 1034
Four replicates from each vial were analyzed. The 2 1042
four replicates from each of the ten blank vials were 2 1048
averaged. These 10 values were averaged again and a 2 1054
standard deviation was calculated. The standard 2 1055
deviation was multiplied by the Student t number for Average 1045
n-1 degrees of freedom (3.25 for n=10), at 99% Standard Deviation 10.5
confidence levels to determine the method detection % CV (precision) 1.0%
limit. The MDL was calculated to be 50 ppb. The % recovery based on 105%
LOQ was calculated by multiplying the MDL by 3. A 1 ppm C (accuracy)
value of 150 ppb was obtained ( see Table 1).
Table 2: Calculated accuracy and precision from ten
replicates of a 1 ppm CIP-100 solution as carbon

Anatel Corporation
2200 Central Avenue, Boulder, CO 80301; Phone (303) 442-5533 Fax (303) 447-8365; E-mail: info@anatel.com; http://www.anatel.com
©Copyright 1999, Anatel Corporation. All Rights Reserved. Printed in USA.
Information, descriptions and specifications are subject to change without notice.
Application Note 12.99.2 December 1999

SWAB RECOVERY

Stainless steel plates were used in the swab recovery test to simulate manufacturing equipment. One side of each
plate was spiked with a solution of active substance or cleaning agent. The plates were allowed to dry completely
overnight at room temperature. A Texwipe alpha swab TX761 was moistened with low TOC (< 25 ppb) water and
the spiked plate surface was swabbed both vertically and horizontally. The swab end was cut off, placed into a vial
to which we added 40-mL of low TOC water. The vial was capped tight, vortexed, and allowed to stand for one hour
prior to analysis. The same volume of each solution that was spiked onto the plates was separately spiked directly
into 40-mL of low TOC water and analyzed. The percent recoveries of the different substances are listed in Table 3.
Reported values are the average of three individual swab samples for each substance. The swab recoveries varied
between 79.3% to 95.9%

Substance ppm C of Spiked ppm C of % Recovery % RSD

Standard Spiked Plate
Solution
CIP-100 1810 1710 94.5 1.8
Sucrose 2663 2112 79.3 4.9
Vancomycin 661 634 95.9 3.0
Endotoxin 902 736 80.0 2.8
Table 3: Representative examples of swab recoveries from cleaning agents and active substances

CONCLUSIONS

This study demonstrates that the Anatel A-2000 Wide-Range TOC Analyzer is suitable for measuring organic
residues on stainless steel surfaces, and that it is a reliable tool for cleaning validation as demonstrated by surface
residue recoveries of 79%-96%. The Anatel TOC analyzer offers low limits of detection, excellent linearity,
precision, and accuracy. All of these TOC results, with the exception of Alconox and Triton-X 100, were generated
using the same TOC method, making this TOC technology a low cost and less time consuming alternative for
cleaning validation.

REFERENCES

1. Food and Drug Administration’s Current Good Manufacturing Practice Regulations; 21 CFR 211.220

2. USP 23 Fifth Supplement; November 15, 1996

Anatel Corporation
2200 Central Avenue, Boulder, CO 80301; Phone (303) 442-5533 Fax (303) 447-8365; E-mail: info@anatel.com; http://www.anatel.com
©Copyright 1999, Anatel Corporation. All Rights Reserved. Printed in USA.
Information, descriptions and specifications are subject to change without notice.