ost pharmaceutical companies in the United States and Europe use laboratory-based total organic carbon (TOC) analyzers to meet USP and EP compendial standards for release of water to manufacturing. Many compa-nies are actively converting, or plan to convert to automated water-release sys-tems using on-line TOC analyzers as the instrument of record. Their motivation is to improve quality and lower the costs of producing pharmaceutical-grade water.

New FDA guidances [1,2] encourage the application of scientifi c and risk-based approaches to the development of automated process control systems in the pharmaceutical industry. The basis of a properly designed and qualifi ed on-line TOC-based real-time release (RTR) system is to identify and understand critical process parameters. Steps required include assessing current knowledge and experience, evaluating the analytical capabilities of available analyzers and analyzing the system design for risks, costs and return on investment [3,4].

Some users of on-line TOC-based real-time water release systems have experienced problems related to on-line TOC results that do not correspond to laboratory-based TOC results, sometimes resulting in factory-wide dysfunction and associated costs. The earliest published report was from Collagen [5], but usually companies do not make these problems public [6]. The cause of these problems can usually be traced to the lack of a proper analytical method validation for the on-line TOC in the actual environment of use. This article will describe methods and criteria that may be useful for validating online TOC instrumentation.

COMPARISON STUDY TEST PLANThere are multiple on-line TOC method-

ologies commercially available, but neither the USP nor the EP compendial TOC chapters reference any particular method. ICH Q7A indicates that on-line TOC methods should be validated and proven suitable under the actual conditions of the pharmaceutical application. The USP and EP TOC chapters suggest that simply passing the system suitability test is suffi cient to qualify the TOC for use in pharmaceutical waters; the documents ICH Q7A, ICH Q2(R1), USP and the AAPS Qualifi cation of Analytical Instruments clearly state otherwise [7,8,9].

By defi nition, a TOC analyzer measures all the covalently bonded organic carbon in solution. Ideally, the TOC should be tested on the organics present in the water, but they are usually not known. Therefore, it was decided to perform an analytical TOC validation of different on-line TOC methodologies. This involved adding known concentrations of compendial organics, as well as organics likely to be present in real-world situations, directly into pharmaceutical-grade water. These controlled standard solutions were then directly introduced into different on-line TOC analyzers to determine their individual recoveries.

APPARATUS AND TEST EQUIPMENTFor the test we used an ultrapure water (UPW) system that produced low levels of TOC (< 5 ppb). The primary water pu-rifi cation system consisted of an activated carbon bed, water softener bed, single-stage reverse osmosis unit, ultra low or-ganic removal bed, UV reactor operating at 184 nm and 254 nm wavelengths and a mixed ion-exchange bed. This make-up water was injected into a storage tank and then recirculated through a second,

larger UV reactor and two larger mixed bed ion-exchange beds to the distribution loop, before going back into the main storage tank. There were multiple points of use connected to the main distribu-tion loop, one of which was used for our testing station.

Most pharmaceutical water systems use storage tanks that are vented to the air through microbial-blocking vent fi lters, thereby adding dissolved CO

2 and O

2 to

the water. To our primary water sample drop stream, we added a commercially available hollow fi ber membrane device [10] to introduce controlled levels of dissolved carbon dioxide and oxygen to simulate this situation. The dissolved CO

2

was used to vary the water conductivity at 25° C from 0.055 μS/cm (deionized) to 1.2 μS/cm. This upper conductivity level matches the USP chapter conductivity limit for Water for Injection (WFI) at 25° C. Since the inlet water was deionized and degassed, we used an on-line conductivity sensor [11] to indirectly measure the CO

2 level and an on-line

dissolved oxygen (DO) sensor [12] to measure the DO of the water.

The system used to add organics, accurately, to a water stream has been previously described [13]. It consists of a NIST-traceable turbine fl owmeter [14] for measuring the water system fl ow rate, a syringe pump and injection tee. Organics were injected into the water stream from a syringe at micro-liter per minute fl ow rates. A computer monitored the turbine meter to dynamically control the syringe’s injection rate of organic to achieve a fi xed dilution ratio. With this system, we were able to introduce controlled organic compound concentrations into multiple on-line TOCs in the analyzer test manifold. From an errors

Robust analytical TOC method validation is essential to the success of any online TOC system, particularly systems that release

pharmaceutical-grade water in real time. Meeting USP 643 or EP 2.2.44 specifi cations may not eliminate risk.

By Jon S. Kauffman PhD., Lancaster Laboratories

analysis, using the injection of a known concentration of NaCl with a precision conductivity measurement, we estimated that the injected organic standard concentration accuracy was within ±3% of the targeted concentration.

TESTED ORGANIC COMPOUNDSTypical laboratory TOC analyzers oxidize all the covalently bonded organic carbon in solution to CO

2, and then measure the

CO2 as carbon mass per solution volume.

Common units of measure are μg of carbon/liter or parts per billion as carbon (ppbC). In order to understand the perfor-mance of on-line TOC analyzers, organics were selected that are not completely defi nitive but may be considered to be indicators of actual performance. Besides

the compendial and diffi cult-to-oxidize organics, we wanted to use organics that were likely to be present in pharmaceuti-cal water. Their actual presence depends on the initial contamination in the raw water, the water purifi cation technologies, the correct operation of the technologies and the extent of the fi nal water purity.

To determine the organics that are likely to be present in purifi ed pharmaceutical waters, we reviewed water purifi cation organic removal articles [15-18, 23], as well as papers published by Dr. Stephan Huber, analyzing pharmaceutical-grade waters using his TOC/liquid chromatographic analyzer [19-20]. Based on these references, we chose to test the organics and test concentrations listed in the Table (below). Chloroform is a disinfection by-product that is

often poorly removed using typical pharmaceutical water purifi cation systems. Additional detailed justifi cations for the selection of these compounds have previously been published [22].

If a TOC analyzer recovers all organics equally well in the actual conditions of use, then one could select the most diffi cult-to-oxidize compound and test its recovery at the compendial TOC limit. This would establish the ability of the TOC analyzer to measure all the TOC. However, it has been observed that the selection of the most diffi cult-to-oxidize organic compounds may depend on the TOC methodology, sample pre-treatment, oxidation system stability and water sample characteristics such as DO, pH and conductivity. A reagentless on-line TOC analyzer using only shortwave UV

light will have diffi cultly oxidizing organics that are optically non-absorptive or that require a lot of oxygen in order to be completely oxidized. The latter factor also depends on the presence of enough available DO in solution to meet the stoichiometric requirements for complete oxidation to CO

2 [22].

TESTED ON-LINE TOC ANALYZERSWe tested commercial versions of three reagent-less on-line TOC analyz-

TABLE – COMPOUNDS TESTEDCompound Concentration Class

Sucrose 500 ppbC Compendial (USP/EP)

1,4 Benzoquinone 500 ppbC Compendial (USP/EP)

Nicotinamide 500 ppbC Diffi cult to oxidize

Methanol 50 and 500 ppbC Simple Alcohol

Isopropanol (IPA) 500 ppbC Simple Alcohol

Acetic Acid 19 ppbC (0.3 μS/cm) Weak Organic Acid

CHCl3 and Sucrose 11 ppbC and 225 ppbC Weak Organic Acid

Trimethylamine 65 ppbC Weak Organic Base

Chloroform 11 ppbC Interference organic

Figure 1. Typical Data from Organic Injection Run

505 ppbC Methanol-High DO-High IC■ “DC/UV” ■ “MC/UV-Persulfate” ■ “DC/UV-Rapid” ■ “MC/UV”

12:14 12:43 13:12 13:40 14:09 14:38 15:070

100

200

300

400

500

600

TOC

pp

bC

ers available for pharmaceutical use. The three analyzers used different TOC methodologies. The fi rst methodology is direct conductometric detection (DC) with partial oxidation of the organics and is referred to as “DC/UV-Rapid” method. The “DC/UV-Rapid” analyzer responds very rapidly to organics in water. It is a reagentless online analyzer. The second methodology uses direct conductometric detection with complete oxidation and is referred to as the “DC/UV” method. The “DC/UV” analyzer, a reagentless online analyzer, has been used in the pharma-ceutical industry since 1990. Two versions of the membrane-conductometric (MC) detector, both with complete oxidation methodology, also were evaluated. The membrane conductometric method separates the CO

2 from the post oxidation

sample into a separate water chamber where the conductivity is measured. The fi rst version is the reagentless membrane conductometric and UV-only oxidation system and is referred to as “MC/UV” method. This is also a reagentless online analyzer. The second version is the membrane conductometric and UV with acid and persulfate reagents and is re-ferred to as “MC/UV-Persulfate” method. The “MC/UV-Persulfate” analyzer is reagent-based and confi gurable for either laboratory or online use. Both of these TOCs have been used on pharmaceutical waters since 1997.

Before testing began, all tested analyzers were required to have factory-issued calibration certifi cates and new UV lamps installed. In addition, each analyzer had to have successfully passed the USP TOC system suitability test.

ORGANIC INJECTION TEST RESULTSFigure 1 shows the graphical results of the response from the four test analyzers for an injection of 500 ppb as carbon of methanol. This injection was done with the DO level greater than 4 ppm as O

2.

This level provides enough DO to exceed the stoichiometric requirements for com-plete oxidation of the organic to CO

2 [22].

The typical CO2 level is about 70 ppb as

carbon or about 0.3 μS/cm of conductiv-ity. This is the case in water systems that have PW storage tanks that are vented to air. For each injection, the average of fi ve consecutive measurements of the water system baseline was subtracted from fi ve consecutive measurements from the center of the TOC peak. The percent recovery was then calculated from the known injec-tion concentration.

ORGANIC PERCENT RECOVERY RESULTSFigure 2 shows the percent recovery results for the test compounds on the dif-ferent TOC analyzers. All the tested TOC instruments recovered 500 ppb of the USP and EP system suitability standards, sucrose and 1,4-benzoquinone, and all analyzers would have passed a USP and EP system suitability test.

The response of the analyzers to the two alcohols showed good percent recovery for all TOC methods, except for the “DC/UV-Rapid,” which showed very low recovery at both concentrations of methanol. Methanol is one of the smallest and simplest organics. Additionally, the percent recovery is not the same for either methanol or iso-propanol (IPA), suggesting a possible non-linear response for these alcohols.

Both “DC/UV” and “DC/UV-Rapid”

showed low recovery of the organic weak acids and bases. Both “MC/UV” and “MC/UV-Persulfate” methods correctly recovered the weak acid and base. Organic nitrogen compounds can be diffi cult-to-oxidize [24]. The diffi cult-to-oxidize nicotinamide (vitamin B3) read correctly on the “MC/UV” and the “MC/UV-Persulfate,” low on the “DC/UV-Rapid” method, but high on the “DC/UV” method.

As reported in the literature [5,24,25] both of the direct conductometric methods showed very high responses to chloroform. “DC/UV-Rapid” result was 136 ppbC and the “DC/UV” result was 346 ppbC to the 11 ppbC chloroform, as shown in Figures 2 and 3. It is believed the oxidation product of chloroform includes hydrochloric acid and with both the DC methods this additional conductivity increases the TOC result. This suggests a possible reason for the direct conductometric method false positive responses to chloroform. The response to the 11 ppbC chloroform for the membrane conductometric analyzers was 21 ppbC for the “MC/UV” and 11.7 ppbC for the “MC/UV-Persulfate” TOC. The 11 ppbC chloroform and 225 ppbC (total TOC of 236 ppbC) sucrose response from the “DC/UV” was 506 ppbC and the “DC/UV-Rapid” response was 313 ppbC. The “MC/UV”

Figure 2. Response of the Different Analyzers in High DO

■ “MC/UV-Persulfate” ■ “MC/UV” ■ “DC/UV” ■ “DC/UV-Rapid”

-50%

0%

50%

100%

150%

200%

250%

300%

505 ppbC Sucrose

500 Benzoquinone

50 ppbC Methanol

500 ppbC Methanol

500 ppbC IPA

19 ppbC Acetic Acid

65 ppbC TMA

499 Nicotinamide

11 CHCl3/225 Sucrose

11 ppbC CHCl3

1250%

3180%

response was 274 ppbC and the “MC/UV-Persulfate” response was 261 ppbC to the expected 236 ppbC injection.

Further research is needed to determine why false positive readings occur with some online TOC analyzers as observed with chloroform, since this problem may result in unnecessary out of specifi cation issues for drug manufacturers. Our results also suggest that false negative readings with weak organic acids and bases warrant further investigation, since under reporting the real TOC value is a possible regulatory issue.

It is important to note that the tested commercial online TOC systems all meet current USP release specifi cations. Given the variability of water sources and pharmaceutical water purifi cation systems, it will be useful to evaluate these TOC technologies thoroughly in combination with the likely organic contamination in the water. These evaluations will ensure their suitability for use in online real-time release of water systems, and minimize potential risks. The USP and EP 2.2.44 suggest that 1,4 benzoquinone is diffi cult to oxidize, and that any TOC analyzer passing the benzoquinone-based system suitability test is suitable for use on pharmaceutical-grade water, but our testing suggests this may not be the case.

It is interesting to note that the 2006 Japanese Pharmacopeia (JP) 15

does not completely harmonize with the USP TOC or EP TOC chapters, and our research, so far supports the conclusion of JP15, which states: “If the apparatus conforms to the apparatus suitability test requirements described in “General Chapter Total Organic Carbon” of the United States Pharmacopeia (USP28, 2005), otherwise described in “Methods of Analysis 2.2.44. Total Organic Carbon in Water for Pharmaceutical Use” of the European Pharmacopoeia (EP 5.0, 2005), the apparatus may be used for monitoring a pharmaceutical water system, provided that water of high purity is used as feed water.

A TOC apparatus, characterized by calculating the amount of organic carbon from the difference in conductivity before and after the decomposition of organic substances without separating carbon dioxide from the sample solution, may be infl uenced negatively or positively, when applied to a sample solution containing ionic organic substances or organic substances comprised of nitrogen, sulfur, or halogens such as chlorine and the like; therefore, the apparatus should be selected appropriately depending on the purity of pharmaceutical water to be measured and on the contamination risk upon apparatus failure [26].” In summary, this says the water must be

of suffi cient purity to use the described TOC apparatus. This description matches that of the direct conductometric TOC method.

References1. Final Report, “Pharmaceutical CGMPs for

the 21st Century – A Risk-Based Approach,”

September 2004, http://www.fda.gov/cder/gmp/

gmp2004/GMP_fi nalreport2004.htm.

2. Guidance for Industry, “PAT – A Framework

for Innovative Pharmaceutical Development,

Manufacturing, and Quality Assurance,”

September 2004, http://www.fda.gov/cder/

guidance/6419fnl.htm.

3. Godec, R., Cohen, N., Automated Release of

Water Using On-Line TOC Analysis and FDA

Risk-Based cGMP, Inspection, and PAT Principles,

Pharmaceutical Engineering, Jan/Feb 2005.

4. Martinez, J. E., On-line TOC Analyzers Are

Underused PAT Tools, BioProcess International,

December 2004.

5. McCurdy, Implementing TOC Testing for USP

23- A Case Study, Pharmaceutical Engineering,

Nov/Dec 1997.

6. Communication with Chris Heiss, CEO of Phar-

maceutical Water Inc., 2850 N. El Paso Street,

Colorado Springs, CO 80907, (719)-475-2100,

email: ch@pharmaceuticalwater.com.

7. From the FDA web site see section XII sub-sec-

tion H at http://www.fda.gov/cder/guidance/

4286fnl.pdf or section 12.8 of Q7A from the ICH

web site: http://www.ich.org/LOB/media/ME-

DIA433.pdf.

8. From the FDA web site www.fda.gov/cder/

guidance/1320fnl.pdf and from the ICH web site

http://www.ich.org.

9. AAPS web site location http://www.aapspharm-

scitech.org/view.asp?art=pt050122 or AAPS

PharmaSciTech 2004; Volume 5, issue 1, Article

22 (http://www.aapspharmscitech.org).

10. Membrana Liqui-Cel web site: http://www.

liqui-cel.com and 2.5 x 8 membrane contac-

tor specifi cation sheet http://www.liqui-cel.

com/uploads/documents/D59_2.5x8Extra-

Flow_Rev12_10-051.pdf.

11. Mettler Toledo Thornton web page http://www.

thorntoninc.com/770max.htm.

12. Swan Analytical Instruments AG Trace Oxygen

Monitor with Faraday Verifi cation SOLO Oxy-

trace web page: http://www.swan.ch/products/

highpuritywater/oxygen.

Figure 3. Chloroform and Sucrose TOC Response

TOC

pp

bC

■ “MC/UV-Persulfate” ■ “MC/UV” ■ “DC/UV” ■ “DC/UV-Rapid”

0

100

200

300

400

500

600

ChCl3 10.9 ppbC

TOC=10.9 ppbC

ChCl3 10.9 ppbC + Sucrose 225 ppbC

TOC=236 ppbC

11.7 21

346

136

261 274

506

313

13. Godec, R., “The Performance Comparison of

Ultrapure Water TOC Analyzers using an

Automated Standard Addition Apparatus.”

Semiconductor Pure Water and Chemicals

Conference, pp. 61-112, 2000.

14. Flow Technology, Inc. turbine fl ow meter web

page: http://ftimeters.com/pages/product/om-

nifl o.html.

15. Gottlieb, M., Meyers, P. “The Production of

Ultra Low TOC Resins”. Semiconductor Pure

Water and Chemicals Conference, pp. 23-42,

1998.

16. Pate, KT;“DI Water Resistivity versus Trace Ion

Levels”. Ultrapure Water, pp. 26- 33, Vol. 8,

No. 1, Jan./Feb., 1991.

17. Mizuniwa, T, Kitami, K, Ito ,M, Miwa ,R.

“Analysis of Organic-combined Chloride,

Sulfate and Nitrate Ions in Ultrapure Water.”

Semiconductor Pure Water and Chemicals

Conference, pp. 111-124, 1999.

18. Governal, RA, Shadman, F. “Design of High-

Purity Water Plants: Fundamental Interac-

tions in Removal of Organic Contamination”.

Ultrapure Water, Sep, 1992.

19. Woiwode, W, Huber, S; “Differnzierende TOC-

Bestimmung zur Charakterisierung von Reinst-

wasser und Ruckstandsprufung im Verlauf der

Reinigungsvalidierung”; Pharm. Ind. 62, 5,

377-381 (2000).

20. Huber, S., DOC-Labor web site: http://www.

doc-labor.de/ and click on USA Flag for English,

click on applications, select pharmaceutical or

mixed bed fi lters or Reverse Osmosis or Anion

fi lter or Resin Examination or Cation Filter

21. Huber, S; personal communication showing an

example of Chromatographic-TOC analysis of

pharmaceutical water.

22. “A Science Based Performance Comparison of

On-Line TOC Analyzers” at www.geinstru-

ments.com.

23. Emery, A.P., Girard JE, Jandik P;”Investigation

of Pure Water Contaminants Stemming From

Ion Exchange Materials,” Ultrapure Water,

Oct. 1988.

24. Rydzewski, J., “Identifi cation of a Critical UPW

Contaminant by Applying an Understanding of

Different TOC Measuring Technologies”, UPW

Watertech Conference Proceedings, Portland,

Oregon, 2001.

25. Chu, Theresa; “Trihalomethanes Can Cause

RO/DI System Problems,” Semiconductor Pure

Water Conference, 1989.

26. Japanese Pharmacopeia 15, page 1602, section

3.5.2 “Monitoring with an Indicator of Total

Organic Carbon (TOC).”

About the AuthorDr. Jon S. Kauffman is the Director of Method

Development & Validation and Biopharma-

ceutical Sciences at Lancaster Laboratories. He

earned a B.S. in Chemistry from Millersville

University; a Ph.D. in Chemistry from Univer-

sity of Delaware; and has been with Lancaster

Laboratories since 1989. His areas of expertise

include mass spectrometry, chromatography, im-

purities testing, extractables and leachables test-

ing, dissolution testing, method validation and

biochemistry. He can be contacted at Lancaster

Laboratories, 2425 New Holland Pike, P.O. Box

12425, Lancaster, PA. 17605-2425, (717) 656-

2308 Ext. 1377, (www.lancasterlabs.com).

Excerpted with permission from Pharmaceutical Manufacturing, November/December 2006.© PUTMAN. All Rights Reserved. On the Web at www.pharmamanufacturing.com.

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