The determination of the oxygen content of ampoules and vials of medical biological preparations filled with an inert gas

Jo~r~l of Bi~t~gi~/ 2¢tan~rdizazion (1985) 13, 87-91

The determination of the o n content of oules and vials of medicM biol cal pre tions filled with an inert g~*

V. G.

khovt and N . S.

The sealing of a m p u l e s and vials containing biological p r e p a ~ t i o n s in an a t m o ~ p h e ~ of nitrogen or other i n e ~ g ~ is currently i n c h i n g in popularity~ This is ~ a u s e the m e t h o d is more ~ o n o m i c a l than vacuum evacuation and considerably fewer ampoules are rejected ~ a result of I ~ k a g e . O n the other hand s ~ l l n g u n d e r nitrogen and argon is associat~ with the risk of a higher oxygen c o n c e n t ~ t i o n in the arm re over the biological pr~Ta~tions and it is therefore ne~-'ssac¢ to estimate the c o n c e n r ~ t i o n of this gas carefully~ The ~0'HO

m ~ s - s ~ c t r o m e t e r s a ~ their complexity ~n:d e x ~ n s e which c o n s i d e ~ b l y decre-*~ their a~'ailability in some manu[acturing e~tablishmenrs. In recent y ~ m S i g w ~ and Schoff 3 have devel a ~ ! a r o g r a p h i c g ~ a n a l y ~ r d e s i g n ~ riot the ~ a l y s i s o f the oxygen content in the atmosphere o f ~ p o u l e s and vials. However this m e r h ~ is comp|icated by c e ~ i n inconveniences, nor l e ~ t ~ i n g the sampling from the a m p u l e s and vials which is carr/e,A our by i m m e r s i n g the containers in water and withdrawing a ~ r t i o n of gas w i t h a s ~ i a l syringe. * Re~:eiv~ for publication 3 Febr'~ry 1 9 ~ . t ~t:~ratoe¢ ~ Biophysics. St=re C o n t . | Institute of M e d k ~ Bio|ogica| P r e F ~ t i ~ s by Siv~e~--Vraiek~ 4 I. ~ t ~ : o w G-2~ US~SR~

ich.

87

V~ G~ PETUKHOV AND N. S. OSIN This p a ~ r describ.s a new m e t h ~ for d e t e r m i n i n g the oxygen content o f a m p u l e s and vials ~ l e d in the a t m o s p h e ~ of an inert gas or nit . T h e sensitivity o f this m e t h ~ is q u i t e sufficient for th e analysis o f the m i n i m a l oxygen c o n c e n t r a t i o ~ m e n d e d by the W H O for biological ~ t i o n s . i T h e high efficiency of t h e device, the technical simplicity and easy operarion r e c o m m e n d it for large scale routine analysis in any laboratory or m a n u f a c t u r i n g esrablishment. T h e description o f the m e t h o d , its f ~ t u r ~ and potential applications ate given below.

p h e n o m e n o n is w e l l - k n o w n in the fieM o f luminescence. It is extremely specific and is induced only by oxygen m o l e c u | ~ , the reduction in th e phosphor~:~cence signal intensity ~ i n g proportional to the log~-ithm o f the n concent~tion. The t e c h n i q u e o f phosp nee m ~ u r e r n e n t is s i m # e but, ~ it is not widely used in m ~ i c i n e or, ~ r r i c u l a H y , in the p r o d u c t i o n o f m e d i ~ l biMogical p r e p a ~ t i o n s , its f~ndamental c o m ~ n e n r s a ~ d ~ c r i ~ +

Electric ¢r)0~¢

To the ~o,.~Jm

~

~P

Fig. L

Diag~mmatic ~ p ~ n t a t i ~ of oxymecer~

~-,e p h o s p h o ~ e n c e is measuroJ by a conventional phosphoroscope w h ich consists of two rotating n o t c h ~ discs I:~tween w h i c h the object u n d er study is placed ( s ~ Fig. I). A ~ u r c e o f lighr on one side o f the p h ~ p h o r o s c o p e e x c i t ~ the phosphora r ~ o r d e r on t he other side. R e d i ~ are on a single ~ i s and ate simultan~ausly driven by a m o t o r so that the notches d o not coincide. ~ e exciting light does not therefore tc~ch the p h o t o t ~ e i v e r w h i c h records only the phosph nee of t h e object which is e m i t t e d after a delay of a ~ u t O-01 s . ) A univee~al dye ~ in y, microbiolo~ atv• virol~y. 1¢ is a mix¢~e of chloride, Le. 3 . 6 ~ i a m i ~ lO*methyi~aridiniu~mand } ~ i a m i r ~ r i d i n e c ~Synonym: fla:¢iP~c~dineh#rochIotlde. ÷

NH~ N* CH~

88

NH z Ct ~

-

HCL

t~IHa HCL

N

NH2,HC/~

02 IN AMPOULES C O N T A I N I N G INERT GAS

On the ~ i s of this p~nciple we ~ v e develop:d the "C~ximerer" for analysing en in amF<~ul~ a n d v i e s c o n t a i n i n g m ~ i ~ l biologic! p ons~ T h e m e t h ~ o f use is follows~ The analyser consists o f t h r e ~ p r i n c i p a l ~ r t s [Fig~ 2(a)]: the p h cot~ with an indicator, p h ce o f w h i c h is sensitive to n; the device for c o n v e y i n g the g ~ from les a n d vials i n t o t h e indicator; a n d an initial v a c u u m p u m p . All thr,.~e d e m e n t s are c o n n e c t e d by a s i n g l e valved v a c u u m line Tr in is used ~ i P J i c a t o r a n d ~ follows. T r i ~ a v i n is dissolved i n a I m i x r u ~ , 0 - 0 0 1 : 1 : 1 by weig,hO, t h e n 0 - 0 5 (by w e i g h t ) h u m a n albumin of al Co. is a d d ~ a n d t h e m i x t u ~ ~ r e d over a fi ~ sill w i t h g ~ i n s o f ~ o u t 0.2--,9"5 r a m . A f t e r d~'ing t h e i n d i c t o r is placed in a quartz g l ~ e a n d covered w i t h a fabric filter the p r i n c i p l e purF~se o f w h i c h is to ~ t a i n t h e i n d i c t o r ~ w d e r in t h e e whe~q v a c u u m e v a c u a t i o n is out. ~e ce o f t h e i n d i c a t o r is e x c i t ~ b y an i n ~ ' a d e ~ e n t l m p w i t h o u t filters. T h e nee is ~ n s e d b y t h e p 4 a o t o m u l t i # i e r , t h e signal is a m # i f i e d and ed. T h e c o n s t r u c t i o n o f a device for t ~ m f e r r i n g t h e gas to t h e i n d i c a t o r d e ~ n d s on t h e t y ~ o f c o n t a i n e r in w h i c h t ~ biological tions ar~ placed, f f t h e c o n t a i n e r is a vim the device is a s i m p l e t h i n r~abber hose e n d i n g w i t h a ermic needle . 2(b)]; if t h e c o n t a i n e r is a n a m p o u l e , t h e device is a gas-tight c o n t a i n e r in w h i c h mecbzanical d e s t n a c t i o n ( b r e a k i n g o f a m p u l e rip) ~ c u ~ [Fig, 2(c)].

~icoto {o)

L~>

Ce)

F/g. 2.

(a) ~ m a t l c

diag~,n d the oxyrneter~ (b) gas evacuation from vials. {c) devi~ for b ~ i n g

89

V. G . P E T U K H O V A N D N . S. O S I N

T h e sensitivi W o f t h e methcM d e ~ n d s on the ratio of t h e ballast v o l u m e o f the v a c u u m s y s t e m o f the d e v i c e in -which the a n a l y ~ d g ~ is d i s t r i b u t e d to the v o l u m e o f the sample. A p p r o x i m a t e ~quivalence o f these volumes ( w h i c h can ~ easily attained technically) mak:es possible the d e t e c t i o n ofO.Ol-O*O005 m l o f oxygen in I I of i n e r t gas. To analyse such concentrations it is e n o u g h ro use a p u m p creating an initi~ vacuum dowt -ira o f m e r c u ~ . T h e calibration o f t h e "Oximeter" is achieved by i n t r o d u c i n g calibrated volumes o f oxygen or air i n t o t h e system. For this pur~3se v a c u u m taps are used by m e a n s o f w h i c h exactly m e ~ u r e d v o l u m e s o f g ~ ate i n t r o d u c e d into a Frevio~Iy evacuated system. Figu~ 3 is a c a l i b ~ t i o n c u r v e s h o w i n g t h e relationship b e t w e e n t h e intensity o f indictor l u m i n e s c e n c e a n d t h e partial oxygen pressure in the system. It can ~ seen in the figure t h a t w i t h i n t h e w i d e ~ n g e o f partial n p ~ s s u r e s the relationship b e a t e n t h e i n t e n s i t y o f i n d i c a t o r ph~osphorescence and the ! o g ~ i t h m o f oxygen c o n c e n t ~ t i o n is linear. T h e oxygen c o n c e n t r a t i o n s covered by this m e t b ~ l range from 2 0 % to 0*02%~

5

0

.........

L~

~50

|

15 P5 POrt~ pressure ~ | 1 ~ }

045

Fig. 3 ~ Relationship ~ t w ~ n the intensity o f phosphorescence of rrlpaflavine and r ~ logarithm of the ~i~l

pr~sure o f oxygen~

~ e m ~ s u r e m e n t t e c h n i q u e d e t ~ n d s on w h e t h e r ~ e d i c a ! biological pre~-rations are s ~ l e d in a vial or in an a m p o u l e . To d e t e r m i n e t h e oxygen c o n ~ n r in vials the needle is i n t r ~ u c e d into t h e r u b b e r r b u t w i t h o u t p i e r c i n g it, so t h a t t h e v a c u u m line o f the device is sealed. W h e n the taps are o ~ n t h e v a c u u m p u m p is s w i t c h e d oil a n d t h e ~¢ initial v a c u u m is created in t h e system, T h e r e u p o n t h e tap is ciogM a n d the n ~ d l e is p i n t o t h e vial w i t h the result t h a t the gas from the vial to the indicator. I f t h e gas d ~ not contain o ~ g e n the intensive o f l u m i n e s c e n c e remains u n c h a n g ~ ; if the gas m i x t u r e in the vial contains o x y g e n t h e i n t e n s i t y o f t h e l u m i n e s c e n c e decreases proportionately t o the l o g a r i t h m o f n c o n t e n t . ~ h e c o n c e n t r a t i o n of t h e o x y g e n can t h e n calculated from t h e calibration curve, T h e prepatatioP~ i n a m p o u l ~ are a n a l y s ~ in a s ~ c i a l gas-tight container. ~ h e n a v a c u u m is crcmted in t h e s y s t e m i n c l u d i n g a c o n t a i n e r w i t h an aml~oule, the tap c o n n o t i n g t h ~ v a c u u m system w i t h t h e p u m p is c l o ~ a n d t h e a m p u l e tip is broken~ ~ e rest o f t h e p ~ e d u r e is ~ ~ o ~ . It s h o u l d ~ n o r m t M r for ~ c h te size a container o f ire size s h o u l d ~ ~ ~ that the ~ l ~ t v o l u m e o f t h e system 90

02 IN AMPOULES C O N T A I N I N G INERT GAS r e m a i n s c o n s t a n t . T h e p h o s p h o r o m e t r i c m e t h ~ m a k e s it possible to carry o u t an analysis in 0"5 r a i n a n d to m e a s u r e t h e o x y g e n c o n c e n t r a t i o n to an accuracy o f at least 5%. T h u s , t h e characteristics o f t h e m e t h o d , the s i m p l i c i t y o f m a n u f a c t u r e and ~ s e ~ o f o ~ r a t i o n o f t h e device c o m m e n d its u ~ in t h e laboratories a n d m a n u f a c t u r i n g e s t a b l i s h m e n t s for t h e e s t i m a t i o n o f n in t h e containers o f m e d i c a l biological preparations. REFERENCES 1. W H O E x e r t Committee on Biological Standardization. Twenty-ninth r e . f t . Wc'HO Tech Rep ~ r I978; 626: 117. 2. Delderfieid AJ, Sutherland IA, Campbell PJ. G ~ analysis of the atmosphere within s~ealed glass a r n ~ u | e s using a q u a d r u p l e ma~s sp~ectrometer~ 3 Biol Stand 1978; 6 : 3 3 1 ~ 3 3 8 . 3~ Sigwart C, Shorf O. Betriebskont~lle der Interbegasung k~ei A m p u | | e n und Ampullenf l ~ h e n ~ Swiss Pharm J I979; 3: 29-32~ 4. Terenin AN. Phoronics o f dye-stuff molecules. : i i r , 1967

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