Application Note 12.99.

3 December 1999

World Leader
in
Total Organic Carbon
Analysis
Model A-2000 Wide Range Total Organic Carbon Analyzer

The Determination of CIP-100 Residues on

Stainless Steel Surfaces By Total Organic Carbon Analysis

INTRODUCTION EXPERIMENTAL CONDITIONS

The purpose of this study was to demonstrate the

performance qualification of the A-2000 TOC
analyzer to measure CIP-100 organic residues on
stainless steel surfaces. CIP-100 is a liquid alkaline
detergent commonly used for cleaning production
equipment in the pharmaceutical and
biotechnology industries. Performance parameters
tested include linearity, method detection limit
(MDL), limit of quantification (LOQ), accuracy,
precision, and swab recovery.
METHODOLOGY
Equipment
• Anatel A-2000 Wide Range TOC Analyzer
with autosampler
• Assorted volumetric flasks and pipets
• Analytical balance
• Texwipe Alpha Swabs TX761
• Eppendorf pipettor (100 µl –1000 µl)
• 40-mL EPA standard vials with a 0.025 mm
teflon septa (Anatel, P/N FG5007501)
• 9” x 13” stainless steel tray, sectioned off into
3” x 3” coupons
Chemicals
• CIP-100 (Steris Corporation)
• Low TOC water, MilliQ or equivalent
(<10 ppb)
• Sodium persulfate and phosphoric acid
(Anatel, P/N FG5011401)
• A-2000 low range TOC calibration kit
(Anatel, P/N FG5010901)
Glassware Preparation
All glassware was cleaned with a dilute solution of
Alconox followed by at least three rinses of MilliQ
water. Glassware was then rinsed with 3%
hydrogen peroxide and followed again with MilliQ
water.
A-2000 TOC Analyzer Calibration
The A-2000 TOC analyzer was pre-calibrated
using Anatel SOP# 2007 “Low Range Multipoint
Calibration Method.” This method involves
preparing and analyzing sucrose TOC standards
ranging from 0 ppb C to 10,000 ppb C to generate
a calibration curve. All subsequent analyses were
performed using this calibration curve.
CIP-100 Linearity Test
A stock standard of CIP-100 was prepared by
weighing 1 gram of CIP-100 into a 1 L volumetric
flask. Solution was diluted to volume with MilliQ
water, and the final weight recorded. Serial
dilutions of the stock standard were prepared as
shown in Table 1.
Key Words
Cleaning Validation
Total Organic Carbon
Method Detection Limit
Precision and Accuracy
Linearity
Swab Recovery

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 12.99.3
Table 1: Dilution scheme for CIP-100 standards
Standard Volume
of
stock (ml)
1 0
2 10
3 5
4 10
5 25
Each standard was transferred to a pre-cleaned
40-mL vial. The vial was rinsed 1 time with the
standard solution. Standards were analyzed for
TOC content. 5 replicates of each standard were
analyzed.
Method Detection Limit (MDL)/
Limit of Quantitation (LOQ)
10 pre-cleaned 40-ml vials were filled with 40-mL
of MilliQ water. One swab was placed in each vial
(tips were cut off). The solution was vortexed and
allowed to sit for 1 hour prior to analysis. Four
replicates of each solution were analyzed.
Accuracy and Precision
A 1 ppm or 1000 ppb as carbon standard was
prepared from the stock standard. The stock
standard was determined to have a 4% carbon
content. Two pre-cleaned 40-mL vials were rinsed
and filled with 40-mL of the 1000 ppb carbon
standard. Five replicates from each vial were
analyzed.
Swab Recovery
Stainless steel trays were sectioned off into 3” x 3”
coupons (8 coupons total per tray). The trays were
pre-cleaned in the same manner as the glassware.
The trays were air-dried immediately prior to use.
Two coupons were used as blanks (no solution
spike). The other six coupons were spiked with
0.5 mL of the desired solution (3 coupons per
concentration). Solutions were prepared to obtain a
0.5 ppm C, 1 ppm C or 5 ppm C spike on the
representative coupon. Spike solution
concentrations were determined as follows:
(extraction vol)(desired TOC level)/application
volume = ppm C in spike solution
For example:
(40-mL)(1 ppm C)/(0.5 mL) = 80 ppm C
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.
December 1999
Total volume Theoretical CIP-100
(ml) concentration (ppm)
0 0
200 25
100 50
100 100
100 250
If CIP-100 is 4% carbon, the concentration of CIP-
100 solution prepared to spike 1 ppm C on the
stainless steel coupon is approximately:
(80ppm C)/0.04 = 2000 ppm CIP-100
For a 1 ppm C coupon spike , a 2000 ppm CIP-100
solution was prepared by weighing out
approximately 1 g of CIP-100 into a 500 mL
volumetric flask. The solution was diluted to
volume with MilliQ water. A 1:2 dilution of the
2000 ppm CIP-100 solution was prepared for a
0.5 ppm C coupon spike. A 10,000 ppm CIP-100
solution was prepared for a 5 ppm C coupon spike
by weighing out approximately 5 g of CIP-100 into
a 500 mL volumetric flask. The solution was
diluted to volume with MilliQ water. 0.5 mL of
each CIP-100 solution was pipetted onto the
middle of the 3” x 3”coupons. The coupons were
allowed to dry completely overnight at room
temperature. Pre-cleaned autosampler vials were
filled with 40-mL of MilliQ water. One swab tip
with handle was placed in each vial and allowed to
soak for approximately 5 minutes prior to use.
Excess water was removed from the swab, by
lightly pressing it against the side of the vial. The
pre-moistened swab was used to “wipe” the dried
coupon as shown.
1 2
3 (turn swab over) 4
Figure 1: Swabbing procedure
Application Note 12.99.3 December 1999

The head of the swab was cut off and placed back
into the 40-mL of water from which it had been
removed.
Two blank coupons on each tray were swabbed in
the same manner. Each swab sample vial was
vortexed and allowed to sit for 1 hour prior to
Figures 1 and 2. The resulting linearity was
excellent as indicated by and R2 value of 0.9997
and a slope of 1 when comparing measured versus
theoretical TOC concentration value.
10000

TOC concentration (ppb)

y = 39.254x + 1.462
8000
analyzing. Four replicates from each swab sample 2
R = 0.9997

vial were analyzed. 0.5 ml of each CIP-100 6000

solution was also pipetted in to another 40-mL vial 4000

and diluted to volume with MilliQ water. Four 2000

replicates of each solution were analyzed. 0
0 50 100 150 200 250

DATA ANALYSIS CIP 100 Concentration (ppm)

• For the linearity test, the measured TOC Figure 1: Concentration of CIP-100 solutions
response values of each CIP-100 solution were versus measured TOC concentration
graphed against the actual CIP-100
concentration and a regression analysis was 10000

Theoretical TOC (ppb)

y = 1.0015x - 33.763
performed. The measured ppb C versus the 8000 2
R = 0.9997
theoretical ppb C was graphed and a second 6000

regression analysis was performed. 4000

• For the method detection limit, the four 2000

replicates from each of the 10 blank vials were 0

averaged together. These 10 values were 0 2000 4000 6000 8000 10000
Measured TOC (ppb)
averaged again and a standard deviation was
calculated. The standard deviation was
multiplied by the Student T number for n-1 Figure 2: Measured TOC concentration versus
degrees of freedom (3.25 for n=10), at 99% theoretical TOC concentration of CIP-100
confidence levels to determine the method solutions
detection limit. The limit of quantitation was
calculated to be the value of three times the A method detection limit was determined to be
method detection limit. 50 ppb C from the analysis of 10 method blank
• The 10 TOC values obtained from the vials. The limit of quantitation was calculated to be
measurement of a 1000 ppb carbon CIP-100 151 ppb C based on the student t test. Results are
standard solution were averaged to determine shown in table 2.
precision and accuracy of the TOC
measurement at this level. Table 2: Calculated average from 10 blank vials
(water+vial+swab)
• % swab recovery was calculated by the
following formula: Vial Number Average TOC (ppb)
1 58
[(ave TOC value from swab vials) –
2 72
(ave blank TOC swab vials) ÷ 3 75
(ave TOC value from spiked vials) – 4 93
(TOC from reagent water)] x 100 = % recovery 5 79
For the purpose of this experiment, the TOC value 6 102
7 60
from the reagent water was assumed to be 10 ppb.
8 83
9 67
RESULTS 10 54
Average 74.3
The TOC values as measured by the A-2000 TOC Standard Deviation 15.5
analyzer for the CIP-100 solutions, and MDL (Student t, n=10) 50 ppb
LOQ 151 ppb
corresponding theoretical TOC values, were
graphed as standard curves and are shown in
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 12.99.3 December 1999

An RSD value of 1% from the analysis of 10 replicates of a 1 ppm C solution of CIP-100, exhibita the precision
that can be achieved using the A-2000 TOC Analyzer. A value of 105% recovery obtained from the same 1 ppm
C solution demonstrates excellent accuracy. (Table 3).
Table 3: Calculated average from ten replicates of a 1 ppm C CIP-100 Solution
Vial Number Measured TOC (ppb)
1 1041
1 1025
1 1039
1 1057
1 1054
2 1034
2 1042
2 1048
2 1054
2 1055
Average 1045
Standard Deviation 10.5
% CV (precision) 1%
% recovery based on 1 ppm C (accuracy) 105%

Mean swab recoveries ranged between 94.5 to 108.3% for the 0.5 ppm C to 5 ppm C spiked coupons
(Tables, 4, 5, 6). These data once again demonstrate the accuracy of measuring CIP-100 residues using the
Anatel A-2000 Wide Range TOC Analyzer.
Table 4: Carbon concentration measured from each coupon
Theoretical Coupon 1 Coupon 2 Coupon 3 Mean RSD
ppb, C ppb, C ppb, C ppb, C ppb, C
500 790 834 800 808 2.8%
1000 1810 1769 1833 1804 1.8 %
5000 7158 7918 6192 6850 7.8%
Table 5: Carbon concentration measured from each spiked solution
Theoretical Replicate 1 Replicate 2 Replicate 3 Mean
ppb, C ppb, C ppb, C ppb, C ppb, C
500 773 761 775 770
1000 1734 1847 1880 1820
5000 6509 6436 6413 6453
Table 6: Percent recovery from swab samples
Solution Conc Coupon 1 Coupon 2 Coupon 3 Mean
(ppb, C) % recovery % recovery % recovery % recovery
770 92.1 97.9 93.4 94.5 ± 4.1
1820 94.8 92.5 96.1 94.5 ± 1.6
6453 109.3 121.1 94.4 108.3 ± 8.3

CONCLUSIONS

The data generated in this experiment demonstrate that the Anatel A-2000 TOC Analyzer is suitable to measure
CIP-100 organic residues on stainless steel surfaces. The method used in this study provides excellent
linearity, precision and accuracy over the concentration range studied, and acceptable recoveries from swab
sampling can be obtained. With the A-2000’s low level detection, quick analysis time and ease of use, TOC
methods can be quickly developed, validated and utilized for pharmaceutical 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.