Analyst, October, 1968, Vol. 93, @. 653-659 653 The Determination of the Di- and Mononitrates of Ethylene Glycol and 1,2-Propylene Glycol in Blood Colorimetric and Gas-chromatographic Methods BY M. H. LITCHFIELD (Imflerial Chemical Industries Ltd., Industrial Hygiene Research Laboratories, Alderley Park, Cheshire) Methods are described for the determination of the nitrate esters, ethylene glycol dinitrate, propylene glycol 1,2-dinitrate, ethylene glycol mononitrate, propylene glycol 1-mononitrate and propylene glycol 2-mononitrate, in blood. These esters can be removed efficiently from blood by extraction with diethyl ether, and determination of the dinitrates can be carried out colorimetrically, after alkaline hydrolysis, by a diazotisation and coupling reaction. As the mononitrates are not determined by this procedure, they do not interfere with the determination of the corresponding dinitrate.Gas chromatography, with the electron-capture detector, provides a sensitive method for detecting and measuring the mononitrates, with adequate separation from the parent dinitrate, and the latter can also be determined by this method as an alterna- tive to the colorimetric procedure. For both methods the effect of possible interferences is investigated, and the efficiency of recovery from blood over the range 0 to 25pg of ester is reported. THE determination of nitrate esters of polyhydric alcohols in explosives, pharmaceuticals and air by colorimetric methods is well e ~ t a b l i s h e d . ~ , ~ ~ ~ , ~ , ~ These methods are relatively non-specific and do not always distinguish between individual nitrates in a mixture of esters.Recent gas-chromatographic procedures, especially those in which the electron-capture detector is used, have largely overcome the latter difficulty, and the identification of nitrate ester mixtures has been a c h i e ~ e d . 6 ~ ~ ~ ~ Modifications of the above methods have been applied to the determination of nitrate esters of polyhydric alcohols in blood and other biological fluids .9 ,lo ,11,12 3 3 For the investigation of the toxicology and metabolism of organic nitrates in experi- mental animals, the non-specific colorimetric methods can be used for the determination of a free ester in blood when other esters are known to be absent. Thus, procedures can be used involving extraction of the blood with an organic solvent, followed by alkaline hydrolysis and colorimetric determination of the liberated nitrite.Although the individual steps of such an analysis are relatively simple, some of the published procedures involve time-consum- ing steps, such as prolonged solvent extraction or removal of solvent, thus requiring careful attention to prevent loss of ester. Of these procedures, that by Hasegawa, Sato, Yoshikawa, Sakabe, Yamaguchi and Hottal3 for the analysis of ethylene glycol dinitrate is the simplest to apply, as it involves a simple extraction of the blood with diethyl ether and does not require any appreciable removal of solvent after the alkaline hydrolysis step and before the colori- metric determination. It is, therefore, suitable for the analysis of large numbers of samples encountered during metabolism studies. No assessment of this method was made regarding the recovery of ethylene glycol dinitrate from blood, the limits of detection, application to other nitrate esters, effects from metabolites in the blood produced during breakdown of ethylene glycol dinitrate or of the interference from normal blood constituents. Part of the present paper is therefore concerned with such an assessment of the method in a modified form, and of its application to the determination of propylene glycol 1,Zdinitrate.It has been demonstrated during metabolism studies that ethylene glycol dinitrate breaks down in blood to give ethylene glycol mononitrate,l* and propylene glycol 1,2-dinitrate to give propylene glycol 1-mononitrate and propylene glycol 2-m0n0nitrate.l~ It is shown here 0 SAC and the author.654 LITCHFIELD : DETERMINATION OF THE DI- AND MONONITRATES OF [Analyst, Vol.93 that these mononitrates are not detected by the colorimetric method described, and the remainder of this paper, therefore, describes a gas-chromatographic method for their deter- mination. As adequate separation of the mononitrates from their parent dinitrates can be achieved on the gas-chromatographic column, this method also provides an alternative way of determining the latter. COLORIMETRIC METHOD REAGENTS- All reagents should be of analytical-reagent grade unless otherwise stated. Diethyl ether. Ethanol, absolute. Sodium hydroxide solution , N . Hydrochloric acid solution, N.Sulphanilic acid solution-Dissolve 10 g of sulphanilic acid in 2 N hydrochloric acid. Warm to dissolve, make up to 1 litre with 2 N hydrochloric acid and filter. Store in an amber-glass reagent bottle. N-l-Naphthylet~yleneaiamine solution-Dissolve 10 g of N-1-naphthylethylenediamine dihydrochloride in distilled water and make up to 1 litre. Filter and store in an amber-glass reagent bottle. Ethylene glycol dinitrate, 1 per cent. wlv in ethanol. Propylene glycol lJ2-dinitrate, 1 per cent. wlv in ethanol. The last two solutions were supplied by I.C.I. Nobel Division. Working standard solutions when required are prepared by diluting 1 ml of the 1 per cent. solution to 100 ml with ethanol so that 1 ml ZE 100 pg of dinitrate; 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20-0 and 25.0ml of this solution are each diluted to 100ml with ethanol and are equivalent to 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0 and 25.0 pg per ml of dinitrate, respectively. PROCEDURE- By pipette, introduce 1.0 ml of blood into a 50-ml separating funnel, followed by 1.0 ml of de-ionised water.Immediately add 10 ml of diethyl ether, gently shake the contents of the funnel for a few seconds and then release the pressure by easing the stopper cautiously. Shake the contents for 1 minute and allow the layers to separate for 4 to 5 minutes. Remove 5ml of the upper ether layer with a pipette and transfer to a stoppered test-tube. Add 7 ml of ethanol, mix, and then add 1 ml of de-ionised water and 0.5 ml of N sodium hydroxide. Mix and heat the solution in a water-bath at a temperature of 40" C for 1 hour.Then pour the contents of the tube into a 25-ml beaker and gently evaporate off some of the ether on a hot water bath at about 50" to 60" C until the volume of liquid remaining is about 9 ml. Add 0.5 ml of N hydrochloric acid, allow the solution to cool, transfer it to a graduated tube and make up to 10 ml with ethanol. Add 1 ml of 1 per cent. sulphanilic acid, allow to stand for 15 minutes and add 1 ml of 1 per cent, N-1-naphthylethylenediamine. After 5 minutes measure the colour in a spectrophotometer at 540 nm in 1 or 4-cm cells against a reference solution prepared from 5 ml of ether and processed in the same way as the sample ether extract. To calibrate the procedure, 1 ml of each of the working standard solutions is added to 5 ml of ether, 6 ml of ethanol are added and the standards then processed in the same way as for the samples, commencing from the addition of 1 ml of de-ionised water in the colori- metric procedure.CALCULATION- By using the above volumes of blood and ether the concentration of dinitrate in the blood, expressed in micrograms per millilitre, equals twice the number of micrograms of dinitrate read from the calibration graph. RESULTS AND DISCUSSION The colorimetric method gives a linear response up to 25 pg of either dinitrate. Propylene glycol 1,2-&nitrate gives a response equivalent to 0.45 of that of ethylene glycol dinitrate. Ethylene glycol mononitrate and the two propylene glycol mononitrates, in amounts up to 100 pg, give no colour when submitted to the procedure, even when the concentration of the sodium hydroxide used is 10 N.October, 19681 ETHYLENE GLYCOL AND l,%PROPYLENE GLYCOL I N BLOOD 655 RECOVERY FROM BLOOD- To 1 ml of blood in a separating funnel, 1 ml of a standard solution of ethylene glycol dinitrate or propylene glycol 1,Zdinitrate in water was added and, immediately after mixing, analysis was carried out by the above procedure.In this way, twelve determinations were carried out at four levels of added dinitrate and the results tabulated (Table I). TABLE I RECOVERY OF ETHYLENE GLYCOL DINITRATE AND PROPYLENE GLYCOL 1 ,%DINITRATE FROM BLOOD Ethylene glycol dinitrate r 3 Recovered, pg Dinitrate & Recovery added, Standard range, Mean deviation per cent. 1 0.98 f0.04 95 to 107 5 4.95 kO.18 94 to 106 A tLg 10 9.75 f0.12 95 to 99 25 24.50 k0-18 97 to 100 Propylene glycol 1,S-dinitrate Recovered, pg & Recovery Mean deviation per cent, 0.97 *0*06 89 to 107 4.90 f0.09 95 to 110 9.70 &O-16 92 to 104 24.20 +0-30 93 to 102 Standard range, Each mean is the result of 12 determinations.LIMIT OF DETECTION- Blank determinations were carried out on twelve samples of rat blood. The mean value of the optical densities thus derived from the blank samples in the 4-cm cells was 0.002, with a standard deviation of 0-002. As the limit of detection has been calculated to be about three times the standard deviation of the blank,16 then the corresponding values for ethylene glycol dinitrate and propylene glycol 1,2-dinitrate equivalent to an optical density of 0.006 are about 0.1 and 0.2 pg per ml, respectively. INTERFERENCES- Three sources of interference are possible : from natural constituents of blood; from mono- nitrates arising from the metabolism of the dinitrates in the b l o 0 d ~ ~ 9 ~ ~ ; and from inorganic nitrite also produced as a result of this metab01ism.l~~~~ It has been shown above that the blood blank is very small and is less than 0.1 pg of ethylene glycol dinitrate and 0.2 pg of propylene glycol 1,2-dinitrate per ml, and thus interference from blood constituents is minimal for concentrations of dinitrate above 1.0 pg per ml. It has also been shown above that the mononitrates do not hydrolyse to nitrite under the conditions of test, and even although they are extracted from blood with ether, as will be shown later, their presence will not influence the result of the colorimetric determination.Inorganic nitrite may be produced in relatively large concentrations in blood (up to 20pg per ml) during in vivo studies of organic nitrates, and it is possible that some may be transferred into the ether layer at the extraction stage and so interfere with the colorimetric determination. To gauge the effect this might have, 1 ml of a standard solution of inorganic nitrite was added to 1 ml of blood and then immediately submitted to the analytical procedure. Six determinations with 25 pg of inorganic nitrite (as NO,-) gave results that did not differ from blood blank values, while six determinations with 50 pg of inorganic nitrite gave a mean result equivalent to 0.25 pg of ethylene glycol dinitrate per ml of blood.It is possible, therefore, that very high concentrations of inorganic nitrite (>25 pg per ml) may interfere with the results for low concentrations of organic dinitrate. STABILITY- Ethylene glycol dinitrate and propylene glycol 1,2-dinitrate are both rapidly broken down by blood to the corresponding mononitrates and inorganic nitrate i!n v ~ ~ Y o . ~ ~ ~ ~ Ether extraction of the blood should be carried out immediately the sample is withdrawn. Once either dinitrate has been extracted into the ether layer, the analysis may be delayed at this stage without fear of loss of dinitrate, provided the extract is stored at 4" to 5" C.656 LITCHFIELD : DETERMINATION OF THE DI- AND MONONITRATES OF [Analyst, Vol. 93 CONCLUSION If present individually, the concentration of the nitrate esters, ethylene glycol dinitrate and propylene glycol 1,2-dinitrate, can be measured in blood samples by a simple procedure involving extraction with ether, alkaline hydrolysis and colorimetric determination by diazo- tisation and coupling. Inorganic nitrite and the mononitrates, ethylene glycol mononitrate, propylene glycol 1-mononitrate and propylene glycol 2-mononitrate, which may be present as a result of metabolism of the dinitrates in blood, do not interfere.A method involving gas - liquid chromatography is now described, in which the mono- nitrates are determined and which, at the same time, provides an alternative means of determining the dinitrates, ethylene glycol dinitrate and propylene glycol 1,2-dinitrate.REAGENTS- GAS-CHROMATOGRAPHIC METHOD All reagents should be of analytical-reagent grade unless otherwise stated. Diethyl ether-Analysis under the gas-chromatographic conditions described below should Sodium sulphate, anhydrous. Nitroglycerine solution, 1 pg per ml in ethanol. Ethylene glycol mononitrate-This was prepared by the method of Ferris, McLean, Marks and Emmonsl7; a 0.1 per cent. w/v solution in ethanol was used. Propylene glycol mononnitrates-The two mononitrates, propylene glycol 1-nitrate and propylene glycol 2-nitrate, were prepared from the corresponding bromohydrins by using the method for ethylene glycol m~nonitratel~; 0.1 per cent. w/v solutions in ethanol were used. Working standard solutions-One millilitre of a 0.1 per cent.mononitrate solution was diluted to 200 ml with ether; 1 ml of solution = 5 pg of mononitrate. When required, 1.0, 2.0, 3.0, 4.0 and 5-0 ml of this solution are each diluted to 25 ml with ether and are equivalent to 0.2, 0-4, 0-6, 0.8 and 1.0 pg of mononitrate per ml, respectively. APPARATUS- An Aerograph 1522 gas chromatograph, with a tritiated foil electron-capture detector and 5-feet x *-inch 0.d. stainless-steel column containing 30 per cent. E301 on 80 to 120-mesh acid-washed Celite, was used. give no peaks corresponding to the nitrate esters tested. OPERATING CONDITIONS- Column temperature, 100" C; injector, 140" C; detector, 135" C; nitrogen flow-rate, 60 ml per minute; and chart speed, 20 inches per hour (Honeywell recorder, 1 mV f.s.d.).Before commencing gas-chromatographic analysis, condition the column by injecting 5 p1 of nitroglycerine solution 1 to 2 hours before the first sample injection (see Results and discussion). PROCEDURE- By pipette, transfer 1.0 ml of blood into a 50-ml separating funnel, followed by 1.0 ml of de-ionised water. Immediately add 10 ml of diethyl ether, gently shake the contents of the funnel and release the pressure by easing the stopper. Shake the contents for 1 minute. Allow the layers to separate for 4 to 5 minutes. Remove as much of the ether layer as possible with a pipette and transfer it to a stoppered cylinder containing about 1 g of anhydrous sodium sulphate. Add 2ml of ether to the remaining contents in the separating funnel, swirl and allow the layers to separate.Remove as much of the ether as possible and add it to that in the stoppered cylinder. Allow the ether extracts to remain in contact with the sodium sulphate for at least 2 to 3 hours and then transfer to a 15-ml calibrated flask. Wash the sodium sulphate with 2 ml of ether, add the washings to the flask and finally make up to volume with ether. Concentrate or dilute the extracts, if necessary, to a known volume of ether and inject 2 p1 into the gas chromatograph. Calibration graphs are prepared by injecting 2-pl portions of the standard solutions in ether and plotting the resultant peak areas against concentration over the range 0.2 to 1.0 pg per ml of nitrate ester.October, 19681 ETHYLENE GLYCOL AND 1 ,%PROPYLENE GLYCOL IN BLOOD CALCULATION- 657 ether volume blood volume' Concentration in blood = concentration in ether extract x RESULTS AND DISCUSSION The sensitivity and reproducibility of the gas-chromatographic responses of the nitrate esters depend on the extent to which the column is conditioned to them.The injection of a nitroglycerine solutionll helps to attain the maximum effect more quickly than by repeated injections of the nitrate ester under test. On a new column more than one injection of nitroglycerine is required before satisfactory sensitivity and reproducibility are attained. Once a column is being used regularly, one conditioning injection of nitroglycerine at the beginning of the day is sufficient. In my experience, the determination of ethylene glycol mononitrate depends on this conditioning effect more than the other esters, The retentions of the five nitrate esters relative to one another on a 30 per cent. E301 -~ ~ ~~ column at 100" C under the conditions of test are given below. Ethylene Propylene Propylene glycol glycol glycol Ester mononitrate 1-mononitrate 2-mononitrate Relative retention 1.00 0.96 1.17 Ethylene Propylene dinitrate 1,2-dinitrate glycol glycol 1.83 2.17 's, Solvent I 2 Solvent 11 L 5 L Injection (a) Injection (b) I.Ethylene glycol 4. Propylene glycol mononitrate 2-mononitrate 2. Ethylene glycol 5. Propylene glycol din itrate I ,Zdinitrate 3. Propylene glycol I -mononitrate Fig. 1. Chromatograms of 2 - 4 injections of mixtures in diethyl ether of: (a) ethylene glycol mononitrate and ethylene glycol dinitrate ; and (b) propylene glycol 1-mononitrate, propylene glycol 2-mononitrate and propylene glycol 1,2-dinitrate Fig.1 shows the chromatograms obtained for the dinitrates compared with the corre- The dinitrates gave an approximately equal response, and this sponding mononitrates.658 LITCHFIELD: DETERMINATION OF THE DI- AND MONONITRATES OF [Analyst, Vol. 93 response is about double that obtained for the mononitrates. The operating conditions quoted in this paper are those required for adequate separation of the two propylene glycol mononitrates, but if this is not required then measurements at higher temperature~l~ will give sharper responses and more sensitivity. RECOVERY FROM BLOOD- It has been shown in the section on colorimetric analysis that the extraction of ethylene glycol dinitrate and propylene glycol 1,2-dinitrate from blood with ether is virtually complete in the range 1.0 to 25.0 pg per ml.At the 0.2 pg per ml level, and with the gas-chromato- graphic finish, recoveries of over 90 per cent. are obtained. The recovery of ethylene glycol mononitrate and the two propylene glycol mononitrates from whole rat blood is shown in Table 11. The recoveries are not as quantitative as for the dinitrates (Table I), presumably because of the relatively increased water solubility conferred by the hydroxyl group present in the mononitrates. TABLE I1 RECOVERY OF ETHYLENE GLYCOL MONONITRATE, PROPYLENE GLYCOL 1-MONONITRATE AND PROPYLENE GLYCOL %MONONITRATE FROM BLOOD Ethylene glycol mononitrate Propylene glycol 1 -mononitrate Propylene glycol 2-mononitrate Mono- nitrate added, Pg 0.5 2.0 5-0 20.0 , 1 Recovered, pg f-h-, Recovery, Standard range, Mean deviation per cent.0.41 f0-02 78 to 92 1.67 f0.08 78 to93 4.15 60.13 78 to87 17-00 f0.65 80 to 90 r 1 Recovered, pg r-A-, Recovery Mean deviation per cent. 0.44 f0.02 84 to 94 1.80 lf0.07 85 to 94 4.44 f0.20 83 to 94 18.80 f0.60 90 to 97 Standard range, Recovered, pg f--A-, Recovery Mean deviation per cent. 0.45 f0.02 86 to 94 1.79 &O-06 85 to 93 4.51 f0-14 87 to 94 18.2 f0-40 88 to 94 Standard range, Each mean is the result of 6 determinations. INTERFERENCE- chromatographic peaks at the retention times of the five esters under review. STABILITY OF THE MONONITRATES IN BLOOD- Although the mononitrates break down relatively slowly in blood in v i t r 0 , ~ ~ 9 ~ 5 it must be remembered that during studies involving ethylene glycol dinitrate and propylene glycol 1,2-dinitrate the latter compounds may be present in the samples, and can rapidly break down to form the mononitrates. Thus, the remarks applied to the dinitrates concerning immediate extraction into ether after sampling apply equally well to the mononitrates.Blood from rats that had not received a dose of organic nitrate ester showed no gas- CONCLUSIONS Ethylene glycol dinitrate, propylene glycol 1 ,e-dinitrate, ethylene glycol mononitrate, propylene glycol l-mononitrate and propylene glycol 2-mononitrate can be determined by gas - liquid chromatography in ether extracts from blood with adequate separation of the mononitrates from the dinitrates, and with no interference from blood constituents. Recovery of the mononitrates from blood is reproducible but slightly less quantitative than for the dinitrates. The skilful technical assistance of Mr. P. B. Banham in the development of the gas- chromatographic method is acknowledged. REFERENCES 1. 2. 3. 4. 5. Akisada, T., Japan Analyst, 1963, 12, 443. Bell, F. K., J . Pharm. Sci., 1964, 53, 752. Sarnoff, E., J . Ass. Off. Agrzc. Chem., 1955, 38, 637. Yagoda, H., and Goldman, F. G., J . Ind. Hyg. Toxicol., 1943, 25, 440. Zurlo, N., and Metrico, L., Medna Lav., 1960, 51, 316.October, 19681 ETHYLENE GLYCOL AND I,%PROPYLENE GLYCOL I N BLOOD 659 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Williams, A. F., and Murray, W. J., Talanta, 1963, 10, 937. Camera, E., and Pravisani, D., Analyt. Chem., 1964, 36, 2108. -- , Ibid., 1967, 39, 1649. Yagdda, H., Ind. Engng Chem. Analyt. Edn, 1943, 15, 27. Ehrner-Samuel, H., in “Proceedings of the 15th International Congress on Occupational Health,” Williams, A. F., and Murray, W. J., Nature, 1966, 210, 816. Zurlo, N., Conti, M., and Nichelatti, T., Medna Lav., 1963, 54, 166. Hasegawa, H., Sato, M., Yoshikawa, H., Sakabe, H., Yamaguchi, M., and Hotta, K., Bull. Natn. Clark, D. G., and Litchfield, M. H., Brit. J . Ind. Med., 1967, 24, 320. -- , in preparation. Roo;, J. B., Analyst, 1962, 87, 832. Ferris, A. F., McLean, K. W., Marks, I. G., and Emmons, W. D., J . Amer. Chem. SOC., 1953, Received May 3rd, 1968 Vienna, 1966, Volume 3, p. 201. Inst. Ind. Hlth., 1962, 8, 10. 75, 4078.