Journal of Chromatography, 396 (I 987) 421-424
`Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
`
`C HROM. 19 503
`
`Note
`
`Assay for epinephrine and its impurities using reversed-phase high(cid:173)
`performance liquid chromatography
`
`Application to control of an eye-drop solution
`
`E. VIDAL-OLLIVIER and G . SCHW AD ROHN
`Laboratoires DULC!S. "Le Mercator", rue de l'fndustrie 98000, Monaco (Monaco)
`and
`C. MAILLARD, G. BALANSARD* and B. OLLIVIER
`Laboratoire de Pharmac:ognosie-Homeopathie. Facultl: de Pharmacie, Universite d'Aix-Marseille, 27 Bou(cid:173)
`levard Jean Moulin, 13385 Marseille CCdex 5 (France)
`(First received October 6th, 1986; revised manuscript received February 13th, 1987)
`
`Epinephrine is a catecholamine used as a sympathomimetic in numerous phar(cid:173)
`maceutical preparations 1, in particular in ophthalmology where it is used to treat
`glaucoma. It is therefore important to develop analytical methods with which to test
`its purity and to determine it. The fact that epinephrine is an unstable molecule
`renders its determination even more impo rtant.
`Epinephrine is easily oxidizable in aqueous solution. This is accentuated by
`the presence of oxygen or heavy metals, by exposure to light and to alkaline pH, or
`by increase in temperature 2 •3 . The final product of oxidation is adrenolutin (Fig. 1).
`Several analytical procedures are used to determine epinephrine: ftuorimetry4 ,
`colorimetry 5, ultraviolet spectrophotometry6 , polarography7
`, gas chromatography 8
`
`-2H
`
`Epinephrine
`
`Epinephrine quinone
`
`l-2H
`
`o~~
`O~N~
`I
`CH3
`
`Adrenoch rome
`
`A<lrenolut in
`
`Vig. I. Oxidation of epinephrine.
`
`0021-9673/ 87 /$03.50 © 1987 Elsevier Science Publishers B. V.
`
`ADAMIS EXHIBIT 1008
`Page 421
`
`

`

`422
`
`NOTES
`
`and high-performance liquid chromatography (HPLC). Reversed-phase HPLC has
`been used to detect epinephrine in injectable solutions9 , epinephrine and norepine(cid:173)
`phrine in intravenous solutions1 0 . The separation of epinephrine and its oxidation
`products has been reported 11 . However, to our knowledge no method has been de(cid:173)
`scribed for simultaneous detection of epinephrine, and of norepinephrine, adrenalone
`and adrenochrome which are possible impurities of epinephrine.
`We thus report a reversed-phase HPLC procedure which enables not only the
`analysis of epinephrine present in an eye-drop solution, but also the detection of
`three possible impurities of epinephrine. Two of these, adrenalone and norepine(cid:173)
`phrine, arise from the methods used to obtain epinephrine. The third, adrenochrome,
`is produced by epinephrine oxidation.
`
`EXPERIMENTAL
`
`A Waters Model 6000 A pump fitted with an U6K universal injector was used
`in combination with an UV M480 spectrophotometer and a Model 833A integrator
`(Merck, Hitachi, France). A data processor was used to calculate retention times and
`peak areas.
`
`('J
`fr•
`
`(:O ~;
`·.CJ
`~ "t. ("-~
`N
`t·':• o;J--
`
`lf )
`
`G • ....
`·-·
`
`co -
`
`10
`
`min
`
`20
`
`Fig. 2. Representative chromatogram of epinephrine (3) in the presence of adrenochrome (1), norepine(cid:173)
`phrine (2) and adrenalone (4). Values at peaks indicate retention times in min.
`
`ADAMIS EXHIBIT 1008
`Page 422
`
`

`

`NOTES
`
`TABLE I
`
`EPINEPHRINE CONTENT OF EYE-DROP SOLUTIONS
`
`423
`
`C.V. = Coefficient of variation.
`
`Sample
`
`Epinephrine
`content
`(g/ 100 ml) (n = 5 )
`
`Eye-drop solution containing I% epinephrine,
`stored for 3 years
`Eye-drop solution containing I% epinephrine,
`stored for 2 years
`Eye-drop solution containing I% epinephrine,
`stored for I year
`
`0.985
`
`0.999
`
`1.031
`
`c.v.
`(%)
`
`1.22
`
`1.22
`
`0.66
`
`Separations were carried out under isocratic conditions using an RT 250-4
`LiChrosorb RP select B 5-µm Hibar column (Merck). The mobile phase was
`methanol- 0.05 M phosphate buffer pH 2 (5:95) containing 0.03 M sodium n~hep­
`tanesulphonate. The flow-rate was 1 ml/min and detection was performed at 220 nm
`at a sensitivity of 0.5 a.u.f.s.
`The standard solutions (10 µl) employed were as follows: epinephrine, 0.5
`mg/ml (Sigma); norepinephrine, 0.25 mg/ ml (Sigma); adrenalone, 0.5 mg/ml (Fluka);
`adrenochrome, 0.5 mg/ml (Siccap-Emmop); epinephrine (0.5 mg/ml) + adrenalone
`(0.5 mg/ml) + adrenochrome (0.5 mg/ml) + norepinephrine (0.25 mg/ml).
`
`Sample
`A 10-µl volume of an eye-drop solution containing 1 % epinephrine and sta(cid:173)
`bilized with isoascorbic acid was diluted 1/20 in distilled water after storage for 1, 2
`and 3 years at room temperature.
`
`RESULTS AND DISCUSSION
`
`A calibration graph was linear for epinephrine concentrations in the range
`0.05- 1 mg/ml (n = 4) with a correlation coefficient of 0.999. For the concentrations
`investigated the coefficients of variation were between 0.79 and l .55%. The limit of
`detection of epinephrine was < 5 µg/ml at a signal-to-noise ratio of 2: 1.
`Fig. 2. shows a chromatogram of epinephrine (retention time 10.78 min) in the
`presence of adrenochrome, norepinephrine and adrenal one whose retention times are
`5.92, 8.01 and 20.18 min respectively. It should be noted that commercial adreno(cid:173)
`chrome is an impure product; in addition to the main peak at 5.92 min there were
`two secondary peaks at 18.94 and 21.04 min.
`
`Eye-drop solution
`Determination of epinephrine in eye-drop solutions was carried out using ex(cid:173)
`ternal calibration (Table 1).
`Fig. 3 shows the chromatogram of an eye-drop solution after storage for 3
`years. At this wavelength (220 nm) there is no interference from other constituents.
`A peak corresponding to isoascorbic acid is observed at 2.77 min.
`
`ADAMIS EXHIBIT 1008
`Page 423
`
`

`

`424
`
`"· , .. __
`
`NOTES
`
`...,
`•.o
`
`~v-
`N <'!
`~") ·J.)
`
`N
`_,
`
`<:1'
`
`ro
`.
`"'"
`......
`-·
`
`N
`":
`,,;.
`
`~
`
`"'
`-s:
`
`•::;;)
`N
`
`0
`
`10
`
`min
`
`20
`
`Fig. 3. Typical chromatogram of an eye-drop solution containing l % epinephrine (1) and stabilized with
`isoascorbic acid(2), stored for 3 years at room temperature. Values at peaks indicate retention times in
`min.
`
`This procedure is rapid, sensitive and reliable. It is thus possible to determine
`epinephrine in an eye-drop solution and detect possible impurities (adrenalone, no(cid:173)
`repinephrine and adrenochrome). The method seems well suited to routine control
`of eye-drop solutions.
`
`REFERENCES
`
`I D. H. Szulczewski and W. H. Hong, Analytical Profiles of Drug Substances, Vol. 7, Academic Press,
`New York, 1978, p. 193.
`2 D. G. Sixmith, W. M. Watkins and G. 0. Kokwaro, J. Clin. Hosp. Pharm., 7 (1982) 205.
`3 R. Bonevski, J. Momirovic-Culjat and L. Balint, J. Pharm. Sci., 67 (1978) 1474.
`4 A . E. Ciarlone, B. W. Fry and R. L. Parker, Microchem. J., 26 (1981) 436.
`5 F. Trublin, H . Robert and A. M. Guyot-Hermann, Bull. Soc. Pharm. Lille, 2-3 (1976) 161.
`6 A. C. Alves, Ann. Fae. Farm., 12 (1952) 41.
`7 J. Henderson and A . Freedberg, Anal. Chem., 27 ( 1955) 1064.
`8 P. Capella and E. Horning, Anal. Chem., 38 (1966) 316.
`9 D. A. Williams, E. Y. Y. Fung and D. W. Newton, J. Pharm. Sci., 71 (1982) 956.
`10 S. M. Waraszkiewicz, E. A. Milano and A. Dirubio, Am. Pharm. Assoc., 70 (1981) 1215.
`11 E. C. Juenge, P. E. Flinn and W. B. Furman, J. Chromatogr., 248 (1982) 297.
`
`ADAMIS EXHIBIT 1008
`Page 424
`
`