GE
Water & Process Technologies
Analytical Instruments
Test Tube Preparation Guidelines for Low
Total Organic Carbon (TOC) Analysis
Purpose
The proposed United States Pharmacopeia XXIII requires
the replacement of the current Oxidizable Substances
Test with Total Organic Carbon (TOC) analysis for both
purified water (PW) and water for injection (WFI). To sup-
port the proposed requirements for measuring TOC in
the laboratory using an auto sampler, it is essential to
minimize and eliminate the background carbon con-
tributed from test tube and sample preparation tech-
niques.
Scope
The scope of this document is designed to assist phar-
maceutical companies in complying with the proposed
specifications for water quality. This document exam-
ines several different test tube and glassware cleaning
methods.
When analyzing samples in test tubes for total carbon,
background contamination can occur from several
different sources. One of the largest contributions of
potential background carbon can come directly from the
water source that is designated for test tube rinsing and
sample preparation. To make this determination, it is
possible to directly measure the total organic carbon
(TOC) level of the source water using an on-line TOC ana-
lyzer such as the Sievers* Model 800 used in this study. If
the water source is a commercially available bottled
water, then the analysis should be made by sampling
directly from the container. If the water source is a labo-
ratory water system, fill a 1 liter cleaned glass flask and
perform the analysis by sampling from the flask. Table 1
illustrates the results obtained at GE Analytical
Instruments using these techniques.
Table 1 Comparison of Different Low-TOC Waters and
Sampling Methods
HPLC grade water 15.76 +/- 0.72 ppb
GEAI** water in a flask 9.13 +/- 0.26 ppb
GEAI** water connected on -line to instrument 2.38 +/- 0.064 ppb
** GE Analytical Instruments
imagination at work
Aftermarket
Application Note
As water is transferred to flasks and tubes, it can easily
become contaminated, as shown by the higher TOC level
in the GE Analytical Instruments (GEAI) water transferred
to a flask. If possible, evaluate the selected type of source
water to demonstrate that it has consistently low TOC.
A second major source of contamination can occur from
the test tube cleaning procedure. To determine the initial
degree of TOC background contamination, use an
aggressive cleaning procedure. A method that is widely
used in the scientific industry to clean laboratory glass-
ware is a chromic acid solution (GEAI Protocol 914-
80005) taken from the United States Pharmacopeia
under Laboratory Glassware Cleaning, <1051>. Test tube
and other glassware cleaned with this procedure will
establish a low level of TOC background contamination.
After obtaining the lowest possible level of background
TOC contamination, then it is prudent to examine milder
cleaning procedures to obtain the same result. The least
corrosive chemical cleaning procedure examined here is
CIP-100 detergent. As an alternative to cleaning agents,
it is possible to clean glassware using a muffle furnace.
The muffle furnace process requires less labor to per-
form, but the initial equipment cost is significant. The sul-
furic acid wash, the muffle furnace, and the CIP-100
detergent wash procedure had comparable results to the
chromic acid wash procedure, as shown in Table 2. An
advantage found for the CIP-100 detergent was that it
requires only 10 rinses, as compared to the 15 or 20 rins-
es required by other cleaning agents. The Alconox labo-
ratory detergent is not recommended as a cleaning
agent for low TOC work.
The test tubes used in Table 2 were challenged with
enough Octoxynol (Triton X-100) to equal a 50 ppm solu-
tion, as carbon, when the tubes were completely filled
with deionized water. The spike solution was allowed to
dry in each tube, and then the various cleaning proce-
dures were performed. A second opinion is to use only
new test tubes for TOC analysis. This option is expensive
 Table 2 Comparison of Different Cleaning Protocols for Test Tube Cleaning An analogous situation exists in the micro bio-
914-80005 914-80007 914-80016 914-80002 914-80015 logical community where bacterial contamina-
Chromic wash Alconox wash Furnace Sulfuric wash CIP-100 Detergent tion is an issue. Here, the concept of aseptic
11.6 ppb 68.8 ppb 15.8 ppb 36.6 ppb 18.8 ppb technique was developed to address cross-
11.1 78.8 14.1 20.9 14.6 contamination issues that are encountered
19.7 41.0 14.0 13.6 15.1 with microbial work.
24.7 45.7 15.7 21.4 16.8
15.6 99.8 15.3 16.2 18.3
18.1 95.2 22.2 23.6 21.9 This concept can, in part, be applied to the
22.5 90.6 24.0 25.8 24.6 preparation of carbon samples. For example,
------ ------ ------ ------ ------ aseptic concepts that are applicable to carbon
17.6 74.3 17.3 22.6 18.59average, ppb
sample preparation are:
±5.2 ±23.6 ±4.06 ±7.5 ±3.6 std dev, ppb
1) To avoid directly handling septa, pipettes,
Table 3 New Tubes Table 4 Pre-Cleaned Tubes Filled the auto sampler needle, and other equipment
Rinsed and Tested and Tested that comes into direct contact with the sample,
15 rinses before filling I-Chem Series 300 Eagle Picher 2) To avoid breathing on the samples as they
VWR Cat #60826-202 VWR Cat #IRT336-0040 Eagle Picher Cat #140 are prepared,
17.8 ppb 18.9 ppb 24.9 ppb 3) To avoid collecting the first few milliliters of a
15.8 18.5 18.4 sample stream, wait until several volumes pass
24.7 20.5 27.6
through and purge the line before collecting a
32.9 24.5 19.9
33.5 17.2 22.7 sample, and
42.2 19.2 19.7 4) To avoid touching the capped septum of test
27.8 average, ppb 19.8 ave, ppb 22.2 ave, ppb tubes when loading the test tubes into an
±10.2 std dev, ppb ±2.5 std dev, ppb ±3.5 std dev, ppb autosampler.

and also labor-intensive because these new test tubes For example, the septa stored in a plastic bag can be
require a 15 rinse preparation step. The TOC values contaminated by fingerprint oils left by a hand reaching
obtained with this method are listed in Table 3. Yet inside. Table 5 shows elevated TOC levels with septa that
another option is to purchase test tubes pre-cleaned by are deliberately contaminated by fingerprints. The right
the manufacturer. The tubes listed here, however, were column shows septa that are handled on both the top
not directly tested by the supplier for TOC, rather they and the bottom surfaces during sample preparation. The
were tested for volatile organic compounds. Thus, there amount of carbon contamination is reflective of the
is not a guarantee maximum level of TOC. These pre- degree of contact with dirty hands or surfaces during
cleaned test tubes were filled with Sievers low organic sample preparation.
deionized water and analyzed on the instrument. The
results are listed in Table 4. Summary
Three areas of sample preparation have been addressed
Table 5 Aseptic Versus Non- The third step in reducing for their potential contribution of background carbon. To
Aseptic Handling of the Septa background carbon con- obtain consistent TOC results with low carbon background
During Sample Preparation tamination is to observe contamination, the water, the test tubes and the sample
Aseptic Non-Aseptic strict sample preparation preparation technique must be carefully monitored.
10.6 ppb 104.4 ppb technique. Handling the
13.8 52.1 reagents and equipment
19.2 66.9 that come into contact
16.4 82.4
with the samples in a spe-
12.6 85.0
17.4 128.7 cial challenge because
15.0/±3.2 ave, 86.6/±27.2 ave, carbon contamination is
std dev, ppb std dev, ppb essentially everywhere.

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