Gas Chromatographic Determination of Residual Ethylene Oxide by Head Space Analysis S. J. Romano, J. A. Renner, and P. M. Leitner Ethicon, Inc., Somerville, N.J. 08876
A specific and sensitive method is described for the determination of ethylene oxide at the ppm level in sterilized materials. The ethylene oxide in the sample is volatilized into the gaseous head space of an enclosed vial. A portion of the gas is analyzed by a previously calibrated gas chromatograph. The method is simpler than other reported gaseous methods since no elaborate gas extraction apparatus is required. It offers significant advantages over liquid extraction of the sample since no interfering solvent peaks are observed, the method is more rapid (50 samples may be analyzed per 8 hours using automatic injection), and the sensitivity is greater (detection limit of 50 ppb). The method is applicable to a variety of materials.
The health industry has for many years used ethylene oxide (EO) as a means of sterilization. Since the sterilizing ability of EO depends upon its toxic effect on living cells, it is important to bring residual EO down to a safe level before the sterile material comes into contact with human tissue ( I ) . Urbanski and Kainz ( 2 ) have reported a sensitive colorimetric method for epoxy compounds, but this method has not been applied to EO residues in the health field. Critchfield and Johnson ( 3 ) developed a colorimetric method for residual EO based on the generation of formaldehyde, but ethylene glycol interfered. Several titrimetric methods for milligram quantities of residual EO have appeared in the literarure (4-6). Hughes e t al. (7) were the first to publish a procedure for the gas chromatographic separation of EO from a gas mixture. Many workers have since reported the use of the sensitive gas chromatographic technique for residual EO analysis (8-12). Spitz and Weinberger ( 1 3 ) described the procedure which has now become the standard analytical method of the health industry. In this method, acetone is used to extract the EO from the sterilized product, and the extract is subjected to gas chromatography. This method is sensitive but not universally applicable since acetone will not extract residual EO quantitatively from all polymeric materials. Mogenhan, et al. ( 1 4 ) have overcome this difficulty by a vacRoberts, E d . . "Infections and Sterilization Problems," Little, Brown & Co., Boston, Mass., 1972. (2) J. Urbanski and G . Kainz, vikrochim. Ichnoanai. Acta, 1965 (l),
(1) R. B.
60. (3) F. E. Critchfield and J. 8. Johnson, Anal. Chem., 29,797 (1957). (4)J . H . Benedict, J. Amer. Oil Chem. SOC.,34, 450 (1957). (5) E. 0.Haenni and R. A. Fulton, Soap Chem. Spec., 35,101 (1959). (6) D. A. Gunther, Anal. Chem., 37, 11723 (1965). (7) K. J . Hughes, R. W . H u r n , and F. G. Edwards, Gas Chromatogr. Int. Symp., EastLansing, Mich., 7959, 171 (1961). (8) N. Adler, J. Pharm. Sci., 54, 735 (1965). (9) R. K . Kulkarni, D. Bartok, D. K. Ousterhout, and F. Leonard, J. Biomed. Mater. Res., 2, 165,(1968). (10) R. K . O'Leary, W. D. Watkins, and W . L. Guess, J. Pharm. Sci., 58, 1007 (1969). (11) D. J. Brown, J. Ass. Offic. Anal. Chem., 53,263 (1970). '(12) B. Warren, J. Pharm. Pharmacol., 23, 1705 (1971). (13) H. D.Spitzand J. Weinberger. J. Pharm. Sci., 60,271 (1971). (14) J. A. Mogenhan, J . E. Whitbourne, and R. R. Ernst., J. Pharm. Sci., 60,222 (1971).
uum extraction method, but the equipment required is complex and the method is cumbersome. Recently a head space gas chromatographic method for polymer powders was reported ( 1 5 ) . However, acetone was used to force the EO out of the polymer, thereby sacrificing sensitivity. Furthermore, acetone contains an impurity which is difficult to remove and has a retention time very close to that of EO. The method reported herein extracts the EO by simply heating the sample in an enclosed vial. The head space gas is then analyzed by gas chromatography. This method offers significant advantages over others since it employs simple apparatus, is more rapid, and its sensitivity is greater. Because samples of known EO levels were not available and could not be made, the accuracy was checked by running paired samples by the head space method and a dimethylformamide (DMF) liquid extraction method.
EXPERIMENTAL Apparatus. A Hewlett-Packard 5750 gas chromatograph with dual flame ionization detectors was used in this investigation. The chromatographic conditions are listed in Table I for the head space method and Table I1 for the D M F method. Hamilton gas-tight syringes of 1 0 0 - ~ 1(No. 1710) and 50-11 capacity (No. 1705) were used for gas sampling. Screw capped vials of 8.8-ml capacity (V-5081, 17 x 60 mm, SGA Scientific) were used as sample containers for the head space method. The cap was drilled and a Teflon (DuPont) lined septum was used to replace the original cap linear so that syringe needles could be inserted into the vial. Two 4-in. hypodermic needles and a short length of Tygon tubing were required in the standardization procedure. A small laboratory oven set at 100 "C was used to heat the samples. Reacti-Vials of 3-ml capacity were used for the DMF method (Reliance Glass Works Inc.). A Hewlett-Packard Model 7670A Automatic Sampler and a n Infotronics Model CRS 204 Integrator equipped with a TTY printer were employed in the later stages of this work to automate the head space method. Reagents. A small lecture bottle of ethylene oxide gas (EO) equipped with a regulator is required as standard (99.7% pure, Matheson Co.). Reagent grade dimethylformamide dried over molecular sieve 4A was used in the D M F method. Procedure. Head Space Method. Procedure for Standardization. A vial was vented by placing a hypodermic needle through the septum, maintaining the point near the top of the vial. A length of Tygon tubing was connected to the vent needle and the end of the tubing submerged in a beaker of water. Another length of tubing was placed onto the EO cylinder regulator and a hypodermic needle connected to the end. The second, or inlet, needle was inserted through the vial septum pushing the point down to the bottom. T h e EO flow was started through the system in such manner that bubbles emerged from the vent tube at the rate of about one per second. The vial was purged for about 15 minutes. This procedure was carried out under a hood. The inlet needle was removed from the vial and the EO gas in the vial allowed to equilibrate to atmospheric pressure. This was done by removing the vent needle from the vial as the last bubble emerged from the vent tube in the beaker. Using the ideal gas law approximation, it could be shown that the concentration of EO in the vial was 1.86 pg/kl. The standard was further diluted by first purging another vial with dry nitrogen for 1 minute. Using the 50-pl gas-tight sy(15) L. A. Zagar, J. Pharm. Sci., 61,1801 (1972)
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
2327
Table I. Gas Chromatographic Conditions for Head Space Method Column: 6 feet X 'ia-inch 0.d. stainless steel, packed with Porapak R, 100-120 mesh (Waters Associates). Temperature: Column oven Injection port Detector
150 "C 250 "C 280 "C
Gas flow: Helium Air Hydrogen
25 cm3/min 450 cm3/min 25 c m 3 / m i n
Attenuation:
10
Sample Size:
x
4
100 gl
-Table II. Gas Chromatographic Conditions for DMF Method Column: 6 feet X '/4-in. o.d., 4 m m i.d. glass packed with Porapak Q-S 100/120 mesh (Waters Associates). Column is conditioned at 21 5 "C. Temperature: Column oven 165 "C for 24 min, then raised to 215 "C to elute DMF. Injection port 250 "C Detector 280 "C Gas flow: Helium Air Hydrogen
45 cm3/min
500 cm3/min 25 cm3/min
Attenuation:
10
Sample Size:
5
x 4
4
280C
Figure 1. Calibration curve for head space method A . Hot syringe dilution technique. 6. Straight injection dilution technique
2501
,ug E 0 / 3 0 m l DMF
Figure 2. Calibration curve for D M F method
2328
ringe, approximately 20 p1 of EO gas was removed from the first vial. The syringe was removed from the vial and, while keeping the needle pointed upward, the plunger was depressed to 2.0 pl. The nitrogen-flushed vial was placed onto the upward syringe needle and the 2.0 pl of EO was injected into the vial. The syringe was not flushed; it was simply removed from the vial immediately. This vial thus contained 3.72 pg of EO. A 100-pl aliquot of the gas from the second standard vial was injected into the gas chromatograph to obtain instrument response. More highly concentrated standards were made by diluting larger aliquots of the pure EO gas from the first vial. Procedure for Samples. A portion of the sample was placed into a screw-capped vial of the same size used in the standardization procedure. The sample size varied from 20 to 200 mg, depending on the level of EO present. The vial was placed in a 100 "C oven and heated for 1.5 minutes. The vial was then removed from the oven. M o s t materials were sampled while hot; however, no difference was found in the peak height of the standard if run hot or cold. A 100-pl sample of the head space gas was injected into the gas chromatograph in duplicate, and the peak heights were averaged, The cap was removed from the vial and the latter was purged for a few seconds with dry nitrogen. The cap was replaced and the heating and injection were repeated. The EO content of the sample was determined using the sum of the average peak heights from the two sample heatings. D M F Method. Procedure for Standardization. A vial of pure EO gas was prepared as described above. A 3.0-mi aliquot of dry D M F was added to a 3-ml Reacti-Vial and capped with a Teflonlined septum. Using a 50-pl gas-tight syringe, a 2-p1 sample of the EO gas was added to the head space above the DMF. The syringe needle was kept dry since a wet syringe does not measure EO gas accurately. The vial was inverted and allowed to stand overnight for complete solution of EO. Standards of higher EO concentration were prepared in the same manner. Duplicate 5.0-pl aliquots of each standard were injected into the gas chromatograph to obtain peak height responses. Procedure for Samples. A sample of the material to be analyzed was weighed into a Reacti-Vial and 3.0 ml of DMF was added. The samples were heated in a 100 "C oven for 1 hour and then removed and cooled to room temperature. Duplicate 5.0-pl aliquots were injected into the gas chromatograph. The EO content of the samples was calculated by referring to the standard curve.
RESULTS AND DISCUSSION Standardization. Head Space Method. Most investigators fail to include a description of their method of preparation of standards. Obtaining small quantities of accurately weighed gas samples can be difficult. For this reason, we decided upon the described method. The ideal gas law approximation was used as a means of determining EO concentration in the standard. This approximation was found to be valid. The calculated concentration was 1.86 pg/pl at 22 "C. When the EO was weighed in a previously calibrated 100-ml flask, the measured value was found to be 1.89 Fglgl. Also, the EO specific volume is reported by the manufacturer as 1.83pg/pl. The dilution procedure of the EO gas standard was found to be a n area of difficulty due to stratification in the syringe barrel. Because of this, dilution in the syringe should not be done unless the needle is plugged and the barrel heated and then cooled to facilitate mixing. Figure 1 shows that both the hot syringe ( A ) and straight injection dilution ( B ) of EO as previously described give comparable results. A cold dilution in the syringe without the aid of mixing leads to a nonlinear and erroneous calibration curve. DMF Method. The standards for EO in D M F solution were allowed to stand for some time (generally overnight) to ensure complete solution of EO. Analysis of the 1-ml head space gas in the Reacti-Vial showed all the EO to be in solution. Heating the standard solutions, as is done with the samples, had no effect on the EO as evidenced by the fact that peak heights before and after heating were identical. Figure 2 shows a calibration curve for the DMF method.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
/1
SAMPLE E.O.
Table I l l . Comparison of DMF and Heat Space Methods EO,ppm Paired sample material
Polyvinyl chloride tubing
Sample
Run
1
a b C
2
a b C
Latex rubber tubing
1
a b C
2
a b C
Polypropylene
1
a b
Polyurethane tubing
1
c a b C
Silicone rubber tubing
1
a b C
Cotton string
1
Experimental polyester
1
a b
a b C
d 2
a b
Head space 27 20 23 66 197 109 17 16 16 17 20 17 538 454 465 14 15 16 6 6 5 32 26 114 108 109 105 223 210
DMF 26 21 22 61 207 115 16 15 15 23 25 24 482 523 449 13 16 15
a 12 8 27 27 104 100 105 108 240 226
STD. EO
FREON 120
_I 0 mins
mins
A B Figure 3. Chromatograms in head space method A . 3.72 kg
EO standard chromatogram at 10 X 4 attenuation, showing only the EO peak. 6. Polyester sample chromatogram showing both the EO and Freon 12 peaks at 10 X 4 attenuation DMF/
IE0
Table I V . Precision of Head Space Method Paired polyester sample No.
Sample half
EO,ppm
Average
1
a
79 81 76 73 63 57 71 66 84 84
80
b 2
a b
3
a b
4
a b
5
a b
75 minutes
Figure 4. Chromatogram in DMF method
60
7.54 p g EO standard in 3 rnl DMF at 10 X 4 attenuation
69 a4
Non-silanized Porapaks resulted in a curve rather than a straight line calibration. Accuracy and Precision. As already mentioned, samples of known EO levels were not available for checking the accuracy of the head space method. The DMF solvent extraction method was developed as an independent sample wcrk-up procedure, so that a direct comparison could be made between samples. Also, since solvent extraction is used as a health industry standard method, it afforded a comparison of methods ( 1 3 ) . Figures 3 and 4 show chromatograms obtained for the head space and DMF methods, respectively. The sample chromatogram in Figure 3 shows a peak for Freon 12 which is used as a diluent for EO in the sterilization process. Samples of various materials which were sterilized by EO were halved. One half was analyzed by the head space method while the other was analyzed by the DMF method. Table I11 shows the results of these analyses. The agreement between the two methods is remarkably good.
The precision of the head space method was checked by running paired polyester halves against each other. Table IV shows these results. The sample-to-sample variation can be seen from the average EO values which range from 60 to 84 ppm even though all samples were taken from the same sterilization lot. For this reason it was necessary to take paired halves in both the precision and accuracy studies. This work shows the method to be precise with an average deviation of 3.2 ppm between paired halves. Extraction Efficiency. One of the major problems involved in solvent extraction techniques presently used throughout the industry is the extraction of all the EO from the sample. DMF either swells or dissolves all the materials used in this study except cotton. Some data on high levels of EO in cotton are shown in Table V and illustrate this problem. In all six cases reported in the present work, the head space method gave higher results than the DMF method, even after three days’ extraction time. This is most likely due to physical absorption of EO on the cotton fibers. The slight increase in EO levels after three days’ extraction indicates that equilibrium has not been reached. However, it is obvious that the system is
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
2329
Table V. Comparison of DMF and Head Space Methods for Cotton at High EO Levels EO. oom
Sample
Head space
DMF day 1
DMF day 3
1 2 3 4 5 6
494 509 41 2 403 471 516
322 232 288 248 336 337
325
251 301 262 354 345
Table VI. Comparison of Acetone Extraction and Head Space Methods for Polyester String EO, ppm
Sample form
Head space
As is Freeze ground
321, 321 ...
Acetone extraction Day 1
Day2
Day3
Day4
18 123
31 136
32 145
173
56
Table V I I . Comparison of Automated and Manual Head Space Methods-Polyester String EO,DPm Paired sample No.
Manual
Automated
1 2 3 4
28 1 355 171 307 244 196 166 164 149
275 309 219 278 223
5 6 7
a 9
200 167 153 197
close to equilibrium and the DMF extraction could never give the levels of EO obtained from the head space method. Further evidence of this behavior was observed with a polyester string when acetone was used as an extractant. Table VI shows these results. The acetone extraction was run both on the sample as is and freeze-ground to increase the surface area. The sample does not dissolve i? acetone. Calculations indicate that it would take approximately 20 days to extract all the EO from the sample if it does not reach equilibrium before that time. When the solution contains undissolved polar solids, the EO partitions between the solid and liquid phases. Hence. if' polar solids are present, a leveling off of the EO concentration with extraction time indicates only that equilibrium has been achieved, and by no means that all the EO has been extracted from the sample. This could be true only if the
2330
partition characteristics of the EO show a strong preference for the solution over the solid. Therefore, if the solvent does not dissolve, or a t least swell the material, one can never be certain that all the EO has been extracted. This is not the case with the head space method since it is an exhaustive extraction technique. A major problem which could arise is the readsorption of EO onto the sample material before the head space gas has been blown off between heatings. This has not been a problem with materials studied in this work. Automation of the Head Space Method. The very clean chromatogram obtained in the head space method lends itself to automated analysis. A Hewlett-Packard Automatic Sampler was attached to the gas chromatograph. The plate covering the sample turntable was removed and an infrared heating lamp was placed approximately 6 inches away from the turntable. The heat produced at the turntable was measured hy a thermocouple and found to be approximately 100 "C. An Infotronics Model CRS204 integrator was used to measure peak areas. Table VI1 shows a comparison of the manual and automated head space methods as run on paired halves of polyester string. In general, the results show good agreement between automated and manual methods. With the automated system, it was possible to run samples unattended. The punch tape obtained from the TTY printer was later fed into a time-shared computer for calculation of results. With this system, it was possible to run 50 samples in an &hour day. Detection Limits. The quantitative detection limit for the head space method is 0.1 ppm based on a 100-p1 injection from a 100-mg sample. The absolute detection limit is 50 ppb based on injecting 2 ml of gas; however, this has not been proved to be quantitative. The detection limit of the DMF method is 5 ppm based on a 5-pl injection from a 100-mg sample. The detection limit of the DMF or any solvent extraction method depends largely on the purity of the solvent used. In the case of DMF, no interfering peaks were observed. This is not so with acetone. Reagent grade acetone has a peak with a retention time almost identical to that of EO. This impurity can sometimes be removed by distillation but reappears with time. Furthermore, it is impossible to inject large amounts of solvents (100 pl) into a gas chromatograph without overloading the column with solvent. This reduces the absolute amount of EO which can be injected with a liquid, resulting in a loss of sensitivity. This is not the case with a gaseous sample where a large injection has little effect on resolution and does not overload the column. ACKNOWLEDGAMENT The authors wish to acknowledge the technical assistance of J. Weinberger of Johnson & Johnson Research, and L. Okolski of Ethicon. Received for review May 3, 1973. Accepted July 6, 1973.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
