New European Pharmacopoeia requirements for Karl Fischer titration...

Freedom of choice of pyridine-free reagents and other improvements

The European Pharmacopoeia (Ph.Eur.) specifies Karl Fischer titration to measure the water content of many solvents, chemicals and other substances.  Historically, and even until very recently, pyridine-containing KF reagents were specified.  This means that for a very long time, the Ph.Eur. did not meet the state-of-the-art in KF technology, since pyridine-free KF reagents have been available and used successfully since 1980.  If one wanted to take advantage of the improved safety and other benefits of pyridine-free reagents, tedious comparison tests had to be carried out with both types of reagents. Sigma-Aldrich, an innovator in pyridine-free KF reagents, supported its customers by conducting over 100 of these comparison tests in the HYDRANAL® laboratory.

However, the newly-released Ph.Eur. 5.7 contains an important change: The use of a pyridine-based KF reagent is no longer compulsory. Chapter 2.5.12 “Water: Semi-Micro Determination” describes in Method A the direct titration of water and in Method B the indirect method. In practice, the direct method, described below, is easier to carry out.


Method A

"Introduce into the titration vessel methanol R, or the solvent indicated in the monograph or recommended by the supplier of the titrant. Where applicable for the apparatus used, eliminate residual water from the measurement cell or carry out a pre-titration. Introduce the substance to be examined rapidly and carry out the titration, stirring for the necessary extraction time.”

Several points are important to elucidate:

1. The titration agent is a matter of choice. If methanol is used in the titration vessel, the titrant of choice is the one-component HYDRANAL Composite.
   
   
2. In our earlier comparison tests, we often recommended the addition of a suitable solubilizing agent for the sample. To accommodate this improvement, the new pharmacopoeia now states in reference to the solvent “…or recommended by the supplier of the titrant.”
   An example is gentamicin sulphate, for which the Ph.Eur. prescribes methanol to be used in the titration vessel. During suitability tests, we found that gentamicin did not fully dissolve during titration, which resulted in erroneously low water content, and afterwards a too-high recovery of added water. The test results thus did not fulfill the requirements of the suitability test. However, when we used a mixture of HYDRANAL Methanol dry and HYDRANAL Formamide dry in the titration vessel, the gentamicin dissolved well and the results fulfilled the requirements of the suitability test. Both documents are available from us upon request.
   
   
3. In addition to the freedom in the choice of titrant, the new Ph.Eur also permits choices in the medium to be used in the titration vessel, specifically “…methanol R, or the solvent indicated in the monograph or recommended by the supplier of the titrant.”
   
   Acetone provides a perfect example. The Monograph for acetone states that pyridine, which is malodorous and toxic, be introduced into the titration vessel. We have performed successfully the requisite suitability test using both pyridine and odorless HYDRANAL KetoSolver, which requires no hazardous declaration, in the titration vessel. These test results are also available on request.
   

Method B

has basically same requirements as Method A, except after addition of the sample an excess of titrant has to be added. The titrant, which is not consumed by the water in the sample, has to be back titrated using a standard with a known amount of water, like HYDRANAL Water in methanol Standard 5.00.

Suitability. The accuracy of the determination with the chosen titrant must be verified for each substance to be examined. The following procedure, given as an example, is suitable for samples containing 2.5-25 mg water.

The water content of the substance to be examined is determined using the reagent/solvent system chosen. Thereafter, sequential known amounts of water R are added in an appropriate form (at least 5 additions) and the cumulative water content determined after each addition.

1. Calculate the percentage recovery
2. Calculate the regression line of the cumulative water
3. Calculate the percentage mean recovery

The reagent/solvent system is considered to be acceptable if:

• The mean recovery is between 97.5 % and 102.5 %
• The slope b is between 0.975 and 1.025 (deviation ± 2.5 %)
• The error e1 and e2 are not greater than 2.5 %

The suitability test with all necessary calculations is not easy to carry out; neither is the preparation of the necessary documentation. To support our HYDRANAL customers, we have put together a test sheet containing all necessary information, including preparation of the titration curve. So far, we have investigated fifteen substances listed in the Ph.Eur. In each case, we used certified HYDRANAL Water Standards for the addition of water in an appropriate form.

Using a reagent with titer 5 for very low water content, which is mostly the preferred titer, we were not always able to meet the limits of acceptance. In these cases, we successfully worked out a second procedure using HYDRANAL Composite 2. A typical example is the procedure for titration of ethyl acetate, which can also be obtained from us on request.


We currently have carried out suitability tests on the following substances:

Acetone (see Table 1)	Glycerol
Citric acid anhydrous	Lactose monohydrate
Dibutyl phthalate	Methanol
Dichloromethane	Olive Oil
Erythromycine	Potassium citrate
Ethyl acetate	2-Propanol
Gentamicin sulphate	di-Sodium hydrogen phosphate dodecahydrate
D(+)-Glucose monohydrate	Trolamine (triethanolamine)

Other Suitability Tests may be requested from here.

Table 1… Suitability test results for acetone. Titration with HYDRANAL-Composite 5 in 30 mL HYDRANAL-KetoSolver (after addition of sample, consecutive known amounts of water are added and determined)

	Sample	Water
		1	2	3	4	5
Sample size (g)	10.0000
Water added (mg)		11.08	11.1	12.02	7.63	6.31
Water found (mg)	11.2420	11.07	11.01	11.87	7.57	6.28
Water content (%)	0.1124
Recovery (%)		99.95	99.16	98.77	99.22	99.45
Mean recovery (%): Slope: Error 1 (%): Error 2 (%):	99.31 0.991 0.88 1.84

The German edition of Ph.Eur. 5, Chapter 4.1.2 Referenzlösung für Grenzprüfungen (reference solutions for limit tests) contains following information:

Reference solution for Micro determination of Water R 1147300

(Commercially available reference solution for coulometric titration of water delivered with a certified water content in an appropriate solvent)
Suitable HYDRANAL reference solutions for coulometric applications are:

HYDRANAL Water Standard 1.00 (1 mg H2O / g = 1000 ppm, exact value stated on CoA)
HYDRANAL Water Standard 0.10 (0.1 mg H2O / g = 100 ppm, exact value stated on CoA)

Lastly, it has to be pointed out that all other HYDRANAL reagents can also be used if the suitability test does meet the requirements.

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