Ethylene oxide residue measuring methods

K.1 Results of interlaboratory evaluation of methods

K.1.1 EO methods

An interlaboratory evaluation was conducted at 13 laboratories using several EO methods (see

References [112], [113] and [114]) on a series of samples with analytical values distributed from about 40 ppm

to about 350 ppm. The estimated total coefficient of variation of the methods is given in Table K.1.

Another interlaboratory evaluation was made, in which each laboratory used the same EO method (see

Reference [89]). Linear regression data were obtained by comparing results obtained in two laboratories for a

series of samples with analytical values distributed from 3,6 ppm to 26 ppm.

The regression equation calculated was: y = 0,04 + 0,904x; correlation coefficient r = 0,974 (p < 0,000 01).

The interlaboratory coefficient of variation of the method was estimated as 4,0 % at 14 ppm EO or 8,3 % at

30 ppm EO in the matrix tested (unpublished data provided by A. Nakamura, H. Kikuchi and K. Tsuji).

Analytical data from samples of three different EO levels were obtained using both the solvent extraction

followed by a headspace gas analysis procedure (see Reference [136]) and the bromination method (see

Reference [89]) in two laboratories. Results were compared using linear regression analysis, which gave the

following regression data y = −0,03 + 1,07x; correlation coefficient r = 0,999. The interlaboratory coefficient of

variation of the K.4.4 procedure was estimated as 4,7 %, 1,8 % and 2,7 % at 12 ppm, 25 ppm and 56 ppm EO

respectively in the matrix tested (see Reference [132]).

K.2 Apparatus and reagents

K.2.1 Apparatus

K.2.1.1 Gas chromatograph, equipped with a flame ionization detector (FID) or an electron capture

detector (ECD) and chart recorder.

NOTE 1 The ECD will not detect EO unless it is first derivatized with hydrogen bromide.

NOTE 2 An electronic integrator is valuable in obtaining reproducible results.

K.2.1.2 Hypodermic needles and polyvinyl chloride tubing, as required for preparing standards.

K.2.1.3 Volumetric glassware, equipped with PTFE-lined septa or PTFE-sealed valves for preparing

standards.

Care should be taken in selecting glassware of an appropriate volume in order to minimize headspace over

the extraction solution or standard solution. When preparing liquid standards or extracts, headspace should

not exceed 10 % of the standard or extractant volume.

K.2.1.4 Micro-syringe, (5 µl or 10 µl capacity) for injecting aliquots of the extract into the gas

chromatograph.

K.2.1.5 Fume hood, to provide adequate ventilation while preparing standards and samples.

K.2.1.6 Analytical balance, capable of measuring to 0,1 mg.

K.2.1.7 Gas regulator, for lecture bottle containing EO.

K.2.1.8 Gas-tight syringes, of 10 µl, 5 µl, 100 µl and 1 000 µl capacities for use in preparing standards

and for injecting headspace gas on to the column of the gas chromatograph.

K.2.1.9 Laboratory oven, capable of heating samples to (100 ± 2) °C.

K.2.1.10 Laboratory oven, capable of heating samples to (37 ± 1) °C.

K.2.1.11 Water bath, capable of maintaining samples at (70 ± 2) °C.

K.2.1.12 Mechanical shaker.

K.2.1.13 Glass headspace vials with PTFE-lined septa, of nominal 20 ml capacity for preparation of

calibration standards.

K.2.1.14 Flat-bottom screw-cap vial, of a size that will accommodate the sample and extraction fluid,

equipped with a PTFE-lined silicone septum and thin PTFE film, used for extraction of EO and reaction of EO

with bromohydrin if applicable.

K.2.1.15 Injection needle, of dimensions 0,65 mm × 25 mm for addition of hydrobromic acid.

K.2.1.16 Millipore filter, of 0,45 µm pore size for filtration of the reaction mixture before chromatography.

K.2.1.17 Refrigerator, capable of maintaining samples between 2 °C and 8 °C.

K.2.2 Reagents

K.2.2.1 Epoxyethane (ethylene oxide), in suitable gas bottle, 99,7 % pure.

K.2.2.2 2-chloroethanol (ethylene chlorohydrin), 99 % assay.

K.2.2.3 1,2-epoxypropane (propylene oxide), reagent grade.

K.2.2.4 Freshly double-distilled hydrobromic acid, prepared as follows:

Distil 100 ml of 47 % hydrobromic acid in the presence of 100 mg tin (II) chloride. Discard the first 25 ml of

distillate and collect the next 50 ml of distillate. Re-distill 50 ml of the distillate in the presence of 50 mg tin (II)

chloride, discard the first 15 ml of distillate and collect the next 20 ml of colourless liquid (bp 125 °C to

126 °C). Store in a glass-stoppered glass container and use within 1 week.

K.2.2.5 Tin (II) chloride (stannous chloride), reagent grade.

K.2.2.6 Water, of purity suitable for GC.

K.2.2.7 Ethanol, of purity suitable for GC.

K.2.2.8 Propanone (acetone), of purity suitable for GC.

K.2.2.9 Dimethylformamide (DMF), of purity suitable for GC.

K.3 Standard preparation

K.3.1 Preparation of ethylene oxide standards

Where required, prepare appropriate standards as described in J.1.

K.3.2 Preparation of ethylene chlorohydrin standards

Where required, prepare ethylene chlorohydrin standards as described in J.2.

K.3.3 Preparation of propylene oxide (PO) standards

Prepare a PO standard by diluting PO in ethanol to provide a solution containing PO at a concentration of

0,5 µg/ml.

K.4 Product extraction

K.4.1 General

Prepare extracts according to the principles described in 4.4.

K.4.2 Extraction to simulate product use

Use water to simulate product use. Perform simulated-use extraction under conditions that provide the

greatest challenge to the intended use.

For example, extract blood-contacting and parenteral devices with water by filling completely or flushing the

blood path or fluid path (whichever is appropriate).

NOTE When filling completely, ensure that no voids remain.

Where it is not possible to fill components of the device that come into contact with the patient, place all, or a

critical and representative portion, of the device in a suitable container and add water to achieve an

appropriate sample/extraction fluid ratio. Exercise caution; take several representative portions of the device

as necessary to ensure confidence in the data derived from small samples of larger devices.

Extract samples for a time equivalent to or exceeding the maximum time for single use, and at temperatures

that provide the greatest simulated challenge, as described in 4.4.6.2. Alternatively, prepare a series of

extracts (a minimum of three is suggested) representing various shorter periods of time and use these

extraction rates to calculate the effects of longer or daily repeated exposure.

If the assay is not performed immediately, decant the extract from the sample and seal in a PTFE-lined

septum-capped vial. The headspace in the vial of any standard solution or extract shall be less than 10 % of

the total volume. The extract can be stored in a refrigerator at (5 ± 3) °C. The analyst should establish the

shelf life and storage time. Take care when using water extraction to assay EO, as EO may convert to EG or

ECH or both during the storage of the aqueous extract (see Reference [35]).

K.4.3 Exhaustive procedure using thermal extraction

Weigh a 1 g sample to the nearest 0,1 mg, place into a capped septum vial, and heat in an oven at an

appropriate temperature for an appropriate amount of time. The time/temperature regimen is relatively

arbitrary. Vary the time to achieve an equilibrium headspace partial pressure of EO.

Remove the vial from the oven. Inject duplicate 100 µl samples of the headspace gas on to the column of the

gas chromatograph, and determine the areas or heights of the EO peaks. Calculate the mean for the duplicate

samples.

NOTE Take care that column packing material is not picked up on the needle during injection. Experience has shown

that testing the hot sample immediately after it has been removed from the oven will result in an error often greater than

20 % because of loss of material from the syringe as it is removed from the vial and its pressure equilibrates to the room

pressure. Some materials resorb EO as the temperature equilibrates to room temperature. Some materials also appear to

resorb the EO completely in the vial if allowed to cool. In the analysis of these materials, samples and standards may need

to be injected on to the column while they are still hot or warm and then purged (as described above) without further

cooling.

Automated headspace analysers are commercially available. However, the technique may be performed

manually.

Remove the cap from the vial under a hood, and purge the vial for 30 s with dry nitrogen. Replace the cap

using a new septum and repeat the heating and injection to exhaustion. Exhaustion is achieved when an

amount of EO is extracted which is less than 10 % of the first extraction or until there is no analytically

significant increase in the cumulative residue levels detected. Calculate the EO in the sample with reference

to the standard curve by summing the EO values obtained for the mean peak area or peak height

measurements made in each of the sample heatings.

K.4.4 Exhaustive extraction with ethanol followed by headspace gas analysis of the ethanol

extract

K.4.4.1 Calibration standards

Prepare EO standards by diluting EO in ethanol to provide solutions containing EO at concentrations of

0,4 µg/ml, 0,8 µg/ml, 1,2 µg/ml, 1,6 µg/ml and 2,0 µg/ml. Prepare a standard containing propylene oxide (PO)

in ethanol at a concentration of 0,5 µg/ml as described in K.3.3. Cool these standard solutions and appropriate

numbers of the special headspace bottles (see Figure K.1) in a dry ice/isopropanol bath, or equivalent.

Transfer appropriate aliquots of each EO standard solution and the same volume of the PO standard solution

to the headspace bottles. Heat the headspace bottles at 70 °C for 30 min and inject duplicate 100 µl to 1 ml

aliquots of the headspace gas from each bottle on to the column of the gas chromatograph. Measure the

height or area of the EO and PO peaks and plot the peak height or peak area ratio (X axis) against the EO

concentration (Y axis) to give a calibration line.

The addition of PO to the EO standards is used as an internal standard to evaluate the accuracy in the

preparation of the EO standards. The plot of the EO/PO ratio against the target EO concentrations of the

standards would ideally result in a linear correlation coefficient, r, of 1,000 and a linear equation of

y = 0,5x + 0. Parameters such as these indicate a perfectly straight calibration line that has a slope of 0,5 with

a y-intercept of zero. A correlation coefficient of 0,999 or better could be used. A linear slope of greater than

K.1.2 ECH methods

An interlaboratory evaluation was conducted for ECH (see Reference [14]). The estimated total coefficient of

variation of the methods was as follows:

⎯ Intralaboratory 7,46 %

⎯ Interlaboratory 10,99 %

These data were obtained for ECH concentrations of about 3,0 ppm to 100 ppm.

0,5 with a y-intercept of zero indicates that all the EO standards were lower than the target concentrations. A

slope of less than 0,5 with a y-intercept of zero indicates that all the EO standards were higher than the target.

Note that calibration lines with a y-intercept greater or less than zero will lead to EO sample results that are

higher or lower than the actual concentration, respectively, especially at lower EO sample concentrations. The

degree of inaccuracy depends on the distance that the y-intercept is from zero. Last, the height or area of the

PO peak itself should remain relatively constant. Fluctuation in the PO peak height or area indicates variability

in the injection volume of the sample into the GC. However, this should not be an issue with the current level

of GC technology.

K.4.4.2 Analysis procedure

Weigh a 5 g (or 0,5 g) sample, cut into small pieces (5 mm long for tubing, 10 mm square for sheet), to the

nearest 0,1 mg and place into a headspace bottle of 100 ml (or 10 ml) capacity. Add 50 ml (or 5 ml) of PO

standard solution (0,25 µg/ml) to the bottle. Cap the bottle, crimp the cap and heat the sealed bottle at 70 °C

for 3 h with gentle shaking. Inject duplicate 100 µl to 1 ml samples of the headspace gas on to the column of

the gas chromatograph and determine the EO/PO peak ratios. Calculate the mean EO content for the

duplicate samples by reference to the calibration line described in K.4.4.1.

Key

1 liquid

2 headspace

3 septum

4 O-ring

5 clamp

Figure K.1 — Special headspace bottle

K.4.5 Exhaustive extraction with solvent

Accurately weigh an approximately 1 g product sample and place it in capped volumetric glassware of the

appropriate volume to minimize the headspace. Transfer 10 ml of the chosen solvent by pipette into the

volumetric flask. Cap the volumetric flask and allow to stand for 24 h at (25 ± 2) °C

These temperatures and times were those used in the study reported by Marlowe[112], [113], [114]. Other

validated temperatures and times may be required to achieve an exhaustive extraction (see K.4.3).

Inject duplicate 1 µl to 5 µl aliquots on to the column of the gas chromatograph. Calculate the EO in the

samples by reference to the standard curve and calculate the mean for the duplicate samples.

K.4.6 Exhaustive extraction with ethanol followed by preparation of the bromohydrin

derivative and chromatography using a gas chromatograph equipped with an ECD

K.4.6.1 Calibration standards

Prepare EO standards by diluting EO in ethanol to provide solutions containing EO at concentrations of

0,4 µg/ml, 0,8 µg/ml, 1,2 µg/ml, 1,6 µg/ml and 2,0 µg/ml. Prepare a standard containing PO in ethanol at a

concentration of 0,5 µg/ml as described in K.3.3. Prepare standard mixtures by mixing equal volumes of each

EO standard solution and the PO standard solution. This plot is done for the same reason as the plot of the

EO/PO ratio versus EO standard concentrations as described in K.4.4.1.

Transfer 1 ml of each standard mixture to a screw-cap vial. Add two drops (ca. 0,015 g) of hydrobromic acid to

the mixture through the septum using an injection needle. Allow the vial to stand for 1 h at room temperature.

Heat the vial for 1 h at 50 °C in a water bath with gentle shaking, then cool to room temperature.

Add 0,02 g sodium bicarbonate to the vial and shake the vial longitudinally for 30 min. Allow the vial to stand

for 10 min. Shake the vial again horizontally for 30 min. Allow the vial to stand for 10 min and centrifuge at

3 000 r/min (50 s−1) for 5 min. Filter the mixture through a small millipore filter 13).

Inject duplicate 1 µl to 5 µl aliquots of each filtrate on to the column of the gas chromatograph to obtain

responses for peak height ratios of ethylene bromohydrin (EBH) vs. propylene bromohydrin (PBH). Prepare a

calibration line by plotting EBH/PBH peak height ratios vs. amounts of EO (µg). This is done in a manner

analogous to that described in K.4.4.1 for EO and PO peaks.

K.4.6.2 Analysis procedure

Use this procedure with standards prepared as described in K.4.4.1.

Cool the PO standard solution (0,25 µg/ml) and a screw-cap vial in a dry ice/isopropanol bath or equivalent.

Transfer 1 ml of the PO standard solution to the vial.

Weigh a 10 mg to 30 mg portion of the sample to the nearest 0,1 mg and place it in the vial.

Add two drops (approximately 0,015 g) of hydrobromic acid to the vial through the septum using an injection

needle. Let the vial stand for 1 h at room temperature then heat the vial for 8 h at 50 °C in a water bath with

gentle shaking. Heat the vial for an additional 16 h at 50 °C in a laboratory oven, then cool to room

temperature.

Add 0,02 g sodium bicarbonate to the vial and shake the vial longitudinally for 30 min. Allow the vial to stand

for 10 min. Shake the vial again horizontally for 30 min. Allow the vial to stand for 10 min and centrifuge at

3 000 r/min (50 s−1) for 5 min. Filter the mixture through a small millipore filter 12).

Inject duplicate 1 µl to 5 µl aliquots of each filtrate on to the column of the gas chromatograph to obtain

responses for peak height ratios of ethylene bromohydrin (EBH) vs. propylene bromohydrin (PBH).

Calculate the mean of the duplicate samples and determine the EO in the sample by reference to the

calibration line described in K.4.4.1.

Since some medical device materials can contain bromide ions (e.g. butylated rubbers), there is the potential

for the formation of EBH as a degradation product of the EO, similar to the formation of ECH from the

presence of chloride ions. Therefore, a portion of the sample should be analysed for the presence of EBH as a

sterilization residue prior to the preparation of the bromohydrin derivative.

K.4.7 Simulated-use extraction for ethylene chlorohydrin using water

Use the procedure described in K.4.2.

K.4.8 Exhaustive extraction for ethylene chlorohydrin using water

Accurately weigh a portion (or the entire sample) of approximately 1 g to 50 g into capped glassware of

appropriate volume to minimize headspace. Transfer sufficient water to cover the sample portion while

ensuring that the container is filled and capped. Allow to stand for 24 h at (25 ± 2) °C. Agitate the container

and contents vigorously on a mechanical shaker for approximately 10 min 14).

Inject duplicate 1 µl to 5 µl aliquots onto the column of the gas chromatograph. Calculate the concentration of

ECH in the sample from either the relative peak area or peak height of the chromatogram when referenced to

the previously generated standard response curve.

K.5 Gas chromatography

K.5.1 General

Select the most appropriate methods and procedures. Use the appropriate chromatographic procedure that

satisfies the requirements listed in Annex A.

Optimization of conditions may be required.

NOTE In order to improve the accuracy of the measurement, and to detect problems with an injection, many

chromatographers use internal standards in their method.

K.5.2 Extraction to simulate product use for the determination of EO or ECH

Inject 1 µl to 5 µl aliquots of the aqueous extract from K.4.2 or K.4.7.

K.5.3 Exhaustive procedure using thermal extraction

Inject 100 µl to 1 ml aliquots of the headspace gas.

K.5.4 Exhaustive extraction with ethanol followed by headspace gas analysis of the ethanol

extract

Inject 100 µl to 1 ml aliquots of the aqueous extract from K.4.4.

K.5.5 Exhaustive extraction with ethanol followed by preparation of the bromohydrin

derivative and chromatography using a gas chromatograph equipped with an ECD

Inject 1 µl to 5 µl aliquots of the aqueous extract from K.4.6.

Ethylene Oxide Sterilization Residuals Detection Method From Iso 10993-7

Publish Time：2024-05-22