`
`1965, 17, 558-565Received March 31, 1965
`
`Research Papers
`
`
`
`Viscosity and stability relations of the
`
`
`system ascorbic acid: water: poly
`
`sorbate 20
`
`J. R. NIXON AND B. P. S. CHAWLA
`
`The viscosity of dispersions of ascorbic acid in solutions of polysorbate 20 has been
`
`
`
`
`
`
`
`
`determined and found to be Newtonian at all concentrations studied. The course of
`
`
`
`
`the oxidation in polysorbate 20 appeared to be by the normal chain reaction, but the
`
`
`
`
`rate at which it occurred was modified by a number of factors. Incorporation
`
`
`
`
`
`
`within the micelle appeared to be responsible for an increase in oxidation rate, but
`
`
`
`
`the high viscosity of concentrated polysorbate 20 solutions, which would affect the
`
`
`
`
`
`diffusion of oxygen to the reaction site caused a much larger reduction in oxidation
`rate.
`
`ANY pharmaceuticals have been prepared in recent years in which
`
`
`
`M
`
`
`
`the medicament has been solubilised by non-ionic surface-active
`
`
`
`
`agents. Aqueous preparations of oil-soluble vitamins have been par
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`
`
`
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`ticularly popular and increased stability to oxidation has been claimed.
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`
`
`
`Previously this department has reported the oxidation of model relatively
`
`
`
`
`
`water insoluble substances in surface-active agents (Nixon, 1958; Mitchell,
`
`
`
`1960; Swarbrick, 1963). It is possible to include the water-soluble
`
`
`
`vitamin ascorbic acid in this type of preparation and we now describe
`
`
`
`
`its oxidative behaviour in the model system ascorbic acid: water: poly
`
`sorbate 20.
`
`Experimental
`
`
`
`Ascorbic acid. Assay (iodometric) 99%. M.p. 190-192°. [�]i>° 2% in
`water + 22°. pH of 2% in water 2·5.
`Polysorbate 20 (Tween, Honeywill-Atlas Ltd). This material complied
`
`
`
`
`
`
`
`with the manufacturer's specification dated October, 1956.
`• 5H20 as a
`
`
`Copper sulphate. Analar. Used at I x I0-4M CuS04
`
`catalyst.
`1921). pH 3·4. Na2HP04/citric acid (Mcllvaine,
`
`
`Buffer solution,
`
`
`
`
`Determination of solubility. The solubility of the ascorbic acid was
`
`
`
`
`determined by equilibration in glass-stoppered flasks immersed in a water
`± O· l O
`
`
`
`• The end-point was taken as the average between an
`bath at 25°
`
`
`Because of the viscosity of high
`
`under-and over-saturated dispersion.
`
`
`
`concentrations of polysorbate 20 it was necessary to warm the flask to 60°
`
`
`
`to speed the equilibration period. This did not affect the quantity solu
`
`
`
`
`
`bilised. In all instances the excess ascorbic acid separated out as crystal
`line material.
`Determination of viscosity. This was measured using a Ferranti
`
`
`
`
`
`
`
`Shirley cone and plate viscometer fitted with an automatic flow curve
`
`
`
`recorder. The viscosity was measured at 25° using either a 4 cm (angle
`
`
`
`
`From the School of Pharmacy. Chelsea College of Science and Technology,
`
`
`Manresa Road, London, S.W.3.
`
`558
`
`ALKERMES EXH. 2009
`Luye v. Alkermes
`IPR2016-1095 & IPR2016-1096
`
`
`
`ASCORBIC ACID: WATER: POLYSORBATE 20
`
`20’. 26") or 7 cm (angle 20’. 25") cone. Flow curves were determined as
`the shear rate was continuously increased from zero up to 1800 sec‘1 and
`then decreased to zero again. The samples were also subjected to
`recycling.
`Measurement of oxygen uptake. The oxidation of the systems was
`followed by means of a Warburg constant volume respirometer at 25° as
`previously described (Carless & Nixon, 1957). The oxygen uptake of
`polysorbate 20 in water was also measured and subtracted from the total
`uptake as a correction.
`Chromatography of oxidised ascorbic acid solutions. The lower layer
`of a butanolzglacial acetic acidzwater system (40: 10:50) was used to
`develop the chromatogram. The Whatman No.
`1
`filter paper was
`equilibrated for 24 hr with the upper layer of the mixture before develop-
`ment. The spots were made visible with ammoniacal silver nitrate and
`the chromatograms were also examined under ultraviolet light.
`
`Results
`
`The presence of polysorbate 20 did not cause any large increase in the
`solubility of ascorbic acid (line AB, Fig. 1) and at high surface-active
`agent concentrations the solubility fell until in the polysorbate 20 itself
`only 5% w/w of ascorbic acid was soluble. None of the dispersions
`
`
`
`°/0 w/w water
`
`FIG. 1. Viscosity and solubility relationships in the system polysorbate 20/ascorbic
`acid/water. Line A—B is the solubility curve of ascorbic acid.
`_
`_
`_
`Vfiscosity
`contours of one phase system.
`- - - - Viscosity contours of equilibrium liquid in
`Contact with excess ascorbic acid.
`
`showed birefringence when examined under polarised light,
`the absence of liquid crystals.
`The viscosity of all the dispersions was Newtonian and did not vary on
`recycling.
`
`indicating
`
`559
`
`
`
`J. R. NIXON AND B. P. S. CHAWLA
`
`The rather complicated viscosity map, produced by connecting similar
`viscosities within both the solubilised and solubilised plus excess solid
`regions, is shown superimposed on the solubility curve in Fig. 1.
`In the binary system polysorbate 20:water there was a very slow
`increase in viscosity to 30% w/w polysorbate 20 after which the increase
`was extremely rapid and reached a maximum of 5-2 poises at 62-8% w/w
`polysorbate 20. The viscosity then fell gradually and polysorbate 20
`itself had a viscosity of 3-81 poises.
`The initial addition of ascorbic acid to any polysorbate 20 : water
`system caused an increase in viscosity. The subsequent behaviour on
`further addition of ascorbic acid depended on the starting concentration
`of polysorbate 20. At concentrations up to about 60% w/w polysorbate
`20 a point was reached where further addition of ascorbic acid caused
`little or no change in the viscosity. This occurred after the addition of
`10-12% w/w of ascorbic acid. From 60-90% w/w polysorbate 20 the
`viscosity of the dispersions continued to increase with addition of ascorbic
`acid until the solubility limit was reached.
`In the heterogeneous region
`of Fig. 1, the viscosity of solutions on the same tie line was, as expected,
`found to be constant. This proved a useful check on solubility data
`which would otherwise have been difficult
`to determine. For initial
`
`concentrations of polysorbate 20 in excess of 90% w/w a third behaviour
`pattern was observed. Here the initial increase of ascorbic acid caused a
`rapid increase in viscosity but on further addition the contours turned
`back upon themselves and the result was a slight fall of viscosity.
`The viscosity of saturated solutions of ascorbic acid in polysorbate 20:
`water exhibited a similar form to the binary polysorbate 20 : water. The
`peak viscosity was 12-3 poises at 68% w/w polysorbate 20.
`The catalysed oxidation of ascorbic acid in water at pH 3-4 and 6-0 was
`a first order reaction. The rate was approximately twice as fast at the
`higher pH but both showed a rapid increase in oxidation rate at ascorbic
`acid concentrations of less than 8% w/w (Table 1).
`
`TABLE 1. OXIDATION or AQUEOUS ASCORBIC ACID
`
`Concentration of
`ascorbic acid % w/w
`
`2-7
`5-95
`8-1
`ll-9
`162
`
`Oxidation rate (ml/kg/hr)
`
`pH 34
`
`7,500
`3,300
`1,950
`1,650
`1,500
`
`pH 6-0
`
`13,600
`5,900
`3,400
`2,900
`2,800
`
`Catalyst: CuSO,.5I-1,0
`
`The induction period of ascorbic acid in polysorbate 20 was much more
`extended than in water alone, and after the induction period a rise to an
`approximately steady oxidation rate occurred. The period of declining
`oxidation rate was also extended.
`
`The oxidation of ascorbic acid in polysorbate 20 is slightly complicated
`due to the slow uptake of oxygen by the polysorbate 20 itself. This
`occurs more rapidly at acid pH, and at high polysorbate 20 concentrations
`
`560
`
`
`
`ASCORBIC ACID: WATER: POLYSORBATE 20
`
`could form a significant proportion of the total oxygen uptake. Fig. 2
`shows the uptake of oxygen by polysorbate 20 and it can be seen that
`although increasing viscosity did initially cause a fall in oxidation rate,
`at high polysorbate 20 concentrations this had increased again and was
`now in excess of dispersions with low viscosity. The method of increasing
`gas:liquid transfer by increasing the shaking rate did not cause any
`noticeable increase in this oxygen uptake.
`
`21.0
`
`200
`
`
`
`
`
`160
`
`Oxidationrate(ml/hr/kgofpolysorbate20) § 1
`
`(poises)
`
`Viscosity
`
`10
`
`20
`
`30
`
`AU
`
`50
`
`EU
`
`70
`
`BE]
`
`90
`
`Concentration of polysorbate 20 (“/0 w/w)
`
`FIG. 2. The oxidation of polysorbate 20. Temperature 25°. Catalyst 1 X 10‘4M
`CuSO4.5H:_.O. pH 3-4.
`Oxidation rate.
`- - -
`- Viscosity.
`
`Before studying the oxidation in relation to the viscosity solubilisation
`diagram (Fig. 1) the effect of catalyst and pH was determined. Saturated
`solutions in 15 and 30% w/w polysorbate 20 were used. With both
`polysorbate concentrations the rate of oxidation increased rapidly with
`
`EFFECT or coppnn CATALYST ON THE RATE or OXIDATION OF:SATURATED
`TABLE 2.
`SOLUTIONS or ASCORBIC ACID
`
`Polysorbate concentration
`%W/w
`
`15
`
`Copper sulphate
`M
`0
`1 X 10"
`5 X 10”“
`1 X 10"
`5 x 10-‘
`
`Steady oxidation rate
`ml/kg/hr
`15
`130
`480
`670
`1,100
`
`15
`0
`220
`1 x 10*‘
`580
`5 x 10"‘
`750
`1 X 10-‘
`S x 10*‘
`1,240
`
`
`30
`
`increasing catalyst concentration, although no linearity was found
`(Table 2). An increase in pH also caused an increased oxidation rate
`except in the region pH 5-6 to 7-2 where a plateau existed (Fig. 3).
`
`561
`
`
`
`J. R. NIXON AND B. P. S. CHAWLA
`
`1.500
`
`3500
`
`2500
`
`(ml/hr/kgofascorbicacid)
`Oxidationrate
`
`
`1500
`
`500
`
`A
`
`5
`
`6
`
`7
`
`8
`
`9
`
`pH
`FIG. 3. The effect of pH on the oxidation of ascorbic acid in polysorb ate 20. Poly-
`sorbate concentration
`30% w/w Temperature
`25°.
`Catalyst
`Ix 10“‘M
`CuSO4.5H2O.
`
`The oxidation of ascorbic acid-saturated dispersions, both catalysed
`and uncatalysed, was studied at pH 3-4. This pH was adopted to prevent
`the catalysed oxygen uptake rate becoming too fast to measure. The
`large increase in the viscosity of concentrated polysorbate 20 dispersions
`had a negligible effect on the oxidation rate of the uncatalysed reaction.
`As the concentration of polysorbate increased, the oxidation rate rose
`slightly, although there was a sharp fall in rate at polysorbate 20 con-
`centrations greater than 90% W/w (Fig. 4).
`
`B00
`
`l.[][]
`
` 600
`
`ZUD
`
`
`
`
`
`Oxidationrate(ml/hr/kgofascorbicacid)
`
`
`
`Viscosity(poises)
`
`10
`
`20
`
`30
`
`L0
`
`El]
`
`6!]
`
`70
`
`80
`
`90
`
`Concentration of polysorbate 20 (% w/w)
`FIG. 4. The oxidation of saturated solutions of ascorbic acid in polysorbate 20.
`Temperature 25°.
`pH 3-4.
`Catalyst
`1 X 10“M CuS04.5H20. ——
`Uncatalysed.
`- - - — Viscosity.
`
`562
`
`
`
`ASCORBIC ACID: WATER: POLYSORBATE 20
`
`The oxidation of the catalysed systems differed considerably. At lower
`polysorbate concentrations, where the viscosity remained almost un-
`changed, the rate of oxidation rose steadily, but once the viscosity started
`to increase, the oxidation rate fell precipitously and reached a minimum
`at around 75% w/w polysorbate 20. Thus, even after the maximum
`viscosity was passed, the oxidation rate showed a further decrease. There
`was little further change in oxidation rate, although the trend was slightly
`upward until dispersions in pure polysorbate were reached, when a further
`sudden fall in oxidation rate occurred. None of these results showed
`
`significant variation on increasing the rate of agitation.
`The presence of even small traces of polysorbate 20 caused a consider-
`able fall in the oxidation rate of the more saturated solutions of ascorbic
`
`acid when compared with the rate of similar aqueous solutions. This
`suggested that the incorporation of ascorbic acid in the polysorbate, and
`the consequent depletion of the water pseudophase, was resulting in a
`measure of protection. At very Iow polysorbate concentrations the
`protection may be due to the normally found effect of surface-active
`agents on the diffusion of oxygen into solutions (Downing, Melbourne
`& Bruce, 1957). To study this effect, a concentration of 32% w/w
`polysorbate 20 was used; this being the highest concentration possible
`before the viscosity commenced its rapid increase, and also because it
`possessed the highest oxidation rate for a saturated solution (Table 3).
`Conversely, if the concentration of ascorbic acid was constant and the
`polysorbate 20 concentration varied, then increased incorporation of the
`acid in the polysorbate would be expected to confer some degree of
`protection on the ascorbic acid as is also illustrated in Table 3.
`
`TABLE 3. OXIDATION RATE or ASCORBIC ACID IN POLYSORBATE 20: EFFECT or
`SATURATION LEVEL
`
`Polysorbate 20 32% w/w
`with ascorbic acid
`% saturation
`
`Rate of oxidation
`ml/kg,’ hr
`
`Ascorbic acid 20% in
`polysorbate 20 % w/ w
`
`Rate of oxidation
`ml/kg/hr
`
`9-9
`26-2
`57-8
`100-0
`
`12,350
`2,579
`1,124
`763
`
`0
`10
`25
`35
`
`1,450
`1,360
`1,100
`850
`
`Catalyst: CuSO4.5H2O
`
`Because of the small quantities of material involved and the uncertain
`interference of the polysorbate 20 with most assays,
`the quantitative
`formation of the oxidation products was not followed. However, a
`number of chromatograms of the oxidising material were made. The
`polysorbate 20 tended to follow the solvent front, but three spots were
`observable with Rf values in the ranges (a) 0-08-0-09;
`(b) 0-33-0-37;
`(C) 053-059. The spot (b) was faint in all instances.
`In one system
`(ascorbic acid 10% w/W, polysorbate 20 30% W/w) a fourth spot was
`detected under ultraviolet light, Rf value 0-80. This was not identified
`and was probably due to impurity. The spots a, b and c are considered to
`correspond respectively to diketogulonic acid, ascorbic acid and dehydro-
`ascorbic acid. The slight variation from the Rf values of Mapson &
`
`563
`
`
`
`J. R. NIXON AND B. P. S. CHAWLA
`
`Partridge (1949) we consider is caused by the polysorbate 20 increasing
`the hydrophiiic property of the material. The large tail of the spots was
`also due to this cause.
`
`Discussion
`
`Mulley (1961) studied the phase equilibria of systems containing non—
`ionic surface-active agents and suggested a general form of ternary phase
`diagram.
`In the present work solid material separated out once the
`solubility limit was reached and, therefore, only the left-hand portion of
`the diagram would apply. No evidence was found of an anisotropic
`liquid crystalline phase, birefringence and non-Newtonian viscosity being
`absent. The polysorbate 20 was probably too hydrophilic to allow for its
`formation. The transfer would appear to be direct from an S1 to an S2
`type micellar distribution (Windsor, 1954). There was no evidence to
`show when this commenced, nor the relative proportions of each micellar
`type at a given concentration. The alternative is that a weak gel structure
`may exist, but no evidence for this was found since the flow curves were
`Newtonian and not shear dependent.
`The increase in viscosity would appear to be associated with the forma-
`tion of S2 type micelles, and the two forms must coexist
`in dynamic
`equilibrium over most of the diagram (Fig. 1). At the two extremes, one
`or other will predominate, the S1 being the main type at low polysorbate
`20 concentrations. Even so, it cannot be assumed that equal quantities
`of the two micellar entities exist at the highest viscosity although it is
`probably the antagonism of the two types which causes the viscosity
`pattern.
`If the viscosity contours are examined, it is seen that the addition of
`ascorbic acid causes very large increases in viscosity in the region where
`Mulley (I961) predicted the presence of a liquid crystalline phase. As
`the ascorbic acid would tend to be solubilised towards the outside of the
`
`S, type micelle rather than in the hydrocarbon interior, the bulk of the
`micelle would be considerably increased and in consequence the relative
`density of packing of the micellar pseudophase.
`In the S2 type micelle the ascorbic acid would be towards the centre
`and the increase in bulk would not be so great, therefore a slight fall in
`viscosity would result as the S2 type began to predominate. This coupled
`with the mutual antagonism of the two micellar types would appear to
`account for the complex viscosity map.
`The mechanism for the oxidation of ascorbic acid in polysorbate 20
`dispersions, as shown by the chromatograms, did not appear to be
`abnormal. However, solubilisation in the polysorbate, the effect of pH
`and the changes in viscosity did modify the rate at which this oxidation
`took place. At pH values above 7-5, where the plateau region had been
`passed and secondary ionisation had commenced, the oxidation rate was
`too fast to be conveniently studied by the methods available, so that pH 6,
`corresponding to complete primary ionisation, and pH 3-4 were used.
`This meant that only one grouping was being attacked and therefore the
`mechanism of oxidation was simpler.
`
`564
`
`
`
`ASCORBIC ACID: WATER: POLYSORBATE 20
`
`Unlike previous studies, the part played by solubilisation in preventing
`oxidation appeared to be small.
`In previous studies the materials were
`far less water soluble and therefore at equilibrium the bulk would be in
`the micelle.
`In the present instance the solubility in the polysorbate 20
`was low when compared with the water solubility and it was in the water
`where most of the oxidation appeared to take place. Except where the
`saturation of the ascorbic acid solution was in the region of 10% or less,
`two effects were apparent.
`Immediately small amounts of polysorbate
`were present, the oxidation rate fell. This could not be due to solubilisa-
`tion and it is suggested that the fall was caused by a reduction in the rate
`of diffusion of oxygen into the system, similar to the elfect of detergents
`on the aeration of sewage effluents (Downing, Melbourne & Bruce, 1957).
`Once micelles formed, the close proximity of the solubilised molecules
`facilitated the continuance of the oxidation chain reaction and until the
`
`It was
`viscosity began to increase rapidly the rate of oxidation rose.
`in this region of low viscosity that the previous studies were made.
`In the region of high viscosity it is probable that the much slower diffusion
`of oxygen to the site of oxidation is the cause of the very low oxidation rate.
`However, an increased shaking rate, which in the event of undersaturation
`with oxygen would normally increase this, produced no effect. Even so,
`from the shape of the oxygen uptake curve, it appeared probable that
`this was the explanation.
`In support of this the uncatalysed oxidation
`showed no drop over the same region and the oxidation rate even rose
`slightly as the viscosity increased, showing that here the system contained
`an optimum amount of oxygen and that gas exchange was fast enough to
`prevent depletion of the system.
`Once the S2 type micellar system predominated and the viscosity began
`to fall again, the systems showed little further change in oxidation rate
`until almost pure polysorbate 20 was reached.
`In this region the water,
`where the bulk of the oxidation appeared to take place, was enclosed as a
`discontinuous pseudophase inside the polysorbate 20 micelle and any
`ascorbic acid dissolved in it was protected from the oxygen by the poly-
`sorbate itself. Once all the water was eliminated, the rate of oxidation
`in the polysorbate itself showed a further sharp fall which suggested that
`the presence of water even in small quantities inside the micelles allowed
`the easier formation of the initiating and propagating free radicles.
`Acknowledgement. The authors would like to thank Dr. J. E. Carless
`for helpful discussions during the preparation of this manuscript.
`
`References
`
`Carless, J. E. & Nixon, J. R. (1957).
`J. Pharm. Pharmacol., 9, 963-973.
`Dowging, A. L., Melbourne, K. V. & Bruce, A. M. (1957).
`J. app]. Chem., 7, 590-
`59 .
`Mapson, L. W. & Partridge, S. M. (1941). Nature, Lond., 164, 479-480.
`Mitchell, A. G. (1960). Ph.D. thesis, University of London.
`Mulley, B. A. & Metcalf, A. D.
`(1961). Paper presented to 21st International
`Congress of Pharmaceutical Sciences, Pisa (1961).
`Nixon, J. R. (1958). Ph.D. thesis, University of London.
`Swarbrick J. (1963). Ph.D. thesis, University of London.
`Windsor, P. A. (1954).
`Solvent Properties of Amphiphilic Compounds, p. 7, London:
`Butterworth.
`
`565