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Differential pulse polarographic determination of the n-nitroso derivative of a tripeptide in pharmaceutical dosage forms
A n a l y t i c a Chimica A c t a , 1 0 7 ( 1 9 7 9 ) 5 9 - - 6 6 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
DIFFERENTIAL PULSE POLAROGRAPHIC DETERMINATION OF THE N - N I T R O S O D E R I V A T I V E O F A T R I P E P T I D E IN P H A R M A C E U T I C A L DOSAGE FORMS
D. G. P R U E * , F. Q. G E M M I L L J R . , a n d R. N. J O H N S O N
A y e r s t Laboratories Inc., 64 ~iaple St., R o u s e s Point, N Y 1 2 9 7 9 (U.S.A.) (Received 18th September 1978)
SUMMARY A m e t h o d is d e s c r i b e d f o r d e t e r m i n i n g a t r i p e p t i d e ( p a r e p t i d e ) i n t a b l e t s a n d c a p s u l e s , w h i c h is b a s e d o n t h e p u l s e p o l a r o g r a p h i c m e a s u r e m e n t o f its N - n i t r o s o d e r i v a t i v e . T h e e l e c t r o c h e m i c a l b e h a v i o r o f t h e d e r i v a t i v e w a s f o u n d t o b e s i m i l a r t o t h a t o f o t h e r Nn i t r o s a m i n e s with regard to the effects o f t e m p e r a t u r e , pH, a n d m e r c u r y pressure. T h e r e d u c t i o n p r o c e s s is i r r e v e r s i b l e a n d c o m p l i c a t e d b y a d s o r p t i o n . O p t i m u m c o n d i t i o n s for p r e p a r a t i o n o f t h e N - n i t r o s o d e r i v a t i v e , w h i c h a p p e a r t o b e a t y p i c a l , are p r e s e n t e d . S p e c t r o s c o p i c d a t a f r o m i.r., m.s., n . m . r , a n d u.v. a n a l y s e s s u g g e s t t h a t t h e d e r i v a t i v e d i f f e r S ' f r o m t h e p a r e n t c o m p o u n d o n l y i n t h a t it h a s a single N - n i t r o s o g r o u p o n t h e p r o l i n e s e g m e n t o f t h e t r i p e p t i d e c h a i n . R e p l i c a t e a s s a y s o n t a b l e t s c o n t a i n i n g 2 m g o f p a r e p t i d e gave excellent precision with a coefficient of variation of 0.37%. C o m p a r i s o n of results o b t a i n e d on capsules containing 2 mg of pareptide and stored at high temperatures showed good c o r r e l a t i o n b e t w e e n t h e p o l a r o g r a p h i c a n d a n h.p.l.c, m e t h o d . R e s u l t s o b t a i n e d w i t h a n u m b e r o f p o s s i b l e d e c o m p o s i t i o n p r o d u c t s i n d i c a t e t h a t t h e p o l a r o g r a p h i c m e t h o d is specific for pareptide.
P a r e p t i d e , L - p r o l y l - N - m e t h y l - D - l e u c y l g l y c i n a m i d e s u l f a t e , has b e e n s y n t h e sized b y a f o u r - c o m p o n e n t c o n d e n s a t i o n p r o c e d u r e [ 1 ] , a n d has b e e n s h o w n t o h a v e p o t e n t i a l clinical a p p l i c a b i l i t y w i t h r e g a r d t o t h e t r e a t m e n t o f P a r k i n s o n ' s disease a n d d e p r e s s i o n [2, 3 ] . P a r e p t i d e itself d o e s n o t possess e l e c t r o c h e m i c a l a c t i v i t y , b u t it c a n b e c o n v e r t e d b y r e a c t i o n w i t h n i t r i t e t o a p o l a r o g r a p h i c a l l y r e d u c i b l e N - n i t r o s o derivative. T h e r e d u c t i o n o f N - n i t r o s a m i n e s at t h e d r o p p i n g m e r c u r y e l e c t r o d e h a s b e e n i n v e s t i g a t e d a n d is g e n e r a l l y w e l l u n d e r s t o o d . In acidic s o l u t i o n , N - n i t r o s a m i n e s u n d e r g o a n irreversible 4 - e l e c t r o n r e d u c t i o n t o t h e c o r r e s p o n d i n g h y d r a z i n e , a n d in basic s o l u t i o n s u n d e r g o a n irreversible 2 - e l e c t r o n r e d u c t i o n t o n i t r o u s o x i d e a n d t h e p r e c u r s o r a m i n e [4, 5 ] . T h e o b j e c t o f this w o r k was t o d e v e l o p a n a n a l y t i c a l m e t h o d w h i c h was sensitive a n d specific f o r p a r e p t i d e in t h e p r e s e n c e o f possible d e c o m p o s i t i o n p r o d u c t s , a n d s u i t a b l e f o r its d e t e r m i n a t i o n in t a b l e t s a n d capsules. T h e m e t h o d p r e s e n t e d h e r e is b a s e d o n a n i t r o s a t i o n r e a c t i o n c a r r i e d o u t u n d e r m i l d c o n d i t i o n s , a n d s u b s e q u e n t d e t e c t i o n o f t h e derivative b y p o l a r o g r a p h y . This a p p r o a c h has b e e n u s e d in s u c c e s s f u l a n a l y s e s f o r o t h e r p h a r m a c e u t i c a l l y
60
important compounds which have a secondary amine functional group, e.g., ephedrine [6] and adrenaline, methylamphetamine and clorprenaline [7]. The electrochemical behavior of IV-nitrosopareptide, conditions for its formation, and characterization of the derivative are also discussed. EXPERIMENTAL Instrumentation
All polarograms were obtained with a Princeton Applied Research polarographic analyzer and recorder (Model 171) equipped with a mechanical drop knocker (Model 172). A Beckman fiber-junction saturated calomel electrode (SCE) was used as the reference electrode and a platinum wire was employed as the auxiliary electrode. The dropping mercury electrode (DME) had the following characteristics (open circuit) in acetate buffer, pH 4.75, m = 5.08 X 1o-4 g s-‘. The following instrumentation was used for characterization of N-nitrosopareptide: Cat-y U.V. spectrcphotometer model 14, Beckman i.r. spectrophotometer model 12, Varian n.m.r. spectrometer model EM-360, and LKB mass spectrometer model 9000.
Pareptide was synthesized [ 1] at Ayerst Research Laboratories, Montreal, Quebec, Canada. For characterization studies, IV-nitrosopareptide solutions were prepared by dissolving 2 g of pareptide in acetic acid 1 + l), then adding 10 ml of 10% sodium nitrite solution and allowing the mixture to stand overnight at room temperature_ The reaction mixture was evaporated to dryness and extracted with acetone. The acetone was removed under vacuum and the resulting oil dissolved in 10 ml of distilled water. The solution was lyophilized to give an amorphous solid. iv-nitroso-L-(--)-proiine was prepared by the method of Lijinsky et ai. [S] . Colorless crystals were obtained (m-p. 104--105°C; [a]g = -187.4”; C = 0.29 in water). All other chemicals were of laboratory-reagent grade.
Analytical
procedure
for tablets and capsules
Weigh out 5, 10 and 15 mg of pareptide reference standard and quantitatively transfer to separate 100-ml volumetric flasks. Dissolve and dilute to volume with acetic acid (I + 19). Pipet 20.0 ml of each standard into separate 25-ml volumetric flasks, add 2.0 ml of aqueous sodium nitrite solution (70 mg ml-‘) and dilute to volume with water. Mix and leave to stand for 60 min. Pipet 5.0 ml of each reaction mixture into separate 50-ml volumetric flasks containing 5-O ml of 2 M ammonium sulfamate solution_ Swirl, leave for 5 min, and dilute each solution to volume with pH 4.75 acetate buffer (0.5 M sodium acetate and 0.5 M acetic acid). Transfer a quantity of standard solution to the polarographic cell and deaerate for 5 min Calibration_
61 with nitrogen. Insert the electrodes and scan from --0.5 V to --1.2 V at 5 mV s -~, w i t h a d r o p t i m e o f 1 s, a c u r r e n t r a n g e o f 2 0 ~ A a n d a p u l s e a m p l i t u d e o f 100 mY. Measure the peak current at --0.83 Y for each standaxd and construct a calibration curve in the conventional manner. Sample preparation. W e i g h o u t t h e e q u i v a l e n t o f 1 0 m g o f p a r e p t i d e f r o m a tablet or capsule powder pool and quantitatively transfer to a 250-ml e x t r a c t i o n b o t t l e . A d d 1 0 0 . 0 m l o f a c e t i c a c i d (1 ÷ 1 9 ) a n d s h a k e o n a m e c h a n i c a l s h a k e r f o r 6 0 rain. C e n t r i f u g e t h e m i x t u r e a n d p i p e t 2 0 . 0 m l o f t h e c l e a r s u p e r n a t a n t l i q u i d i n t o a 2 5 - m i v o l u m e t r i c flask. P r o c e e d w i t h t h e n i t r o s a t i o n r e a c t i o n as d e s c r i b e d a b o v e . C a l c u l a t e t h e s a m p l e s t r e n g t h f r o m t h e e q u a t i o n : m g / t a b l e t ( o r c a p s u l e ) = U × (Wa/Ws}, w h e r e U = m g o f p a r e p t i d e c o r r e s p o n d i n g t o t h e m e a s u r e d p e a k c u r r e n t , Wa = a v e r a g e t a b l e t o r c a p s u l e w e i g h t (rag), a n d Ws = s a m p l e w e i g h t ( m g ) . It should be noted that N-nitrosamines may have carcinogenic properties, so that due precautions should be taken, RESULTS AND DISCUSSION
Polarographic study T h e N - n i t r o s o d e r i v a t i v e o f p a r e p t i d e gives a single w e l l - d e f i n e d p o l a r o g r a p h i c w a v e in p H 4 . 7 5 a c e t a t e b u f f e r w i t h a p e a k p o t e n t i a l o f - - 0 . 8 3 V vs. S C E . T h e d i f f e r e n t i a l p u l s e c u r r e n t i n c r e a s e s l i n e a r l y w i t h c o n c e n t r a t i o n in t h e r a n g e 0 . t ~ 4 0 ug m l - ' ( T a b l e 1). A t c o n c e n t r a t i o n s a b o v e 4 0 u g m l -~, t h e r e is a c h a n g e i n t h e s l o p e o f t h e c u r v e a n d a s e c o n d l i n e a r r a n g e is o b s e r v e d . T h e E ~/2 v a l u e s r e m a i n e s s e n t i a l l y u n c h a n g e d in t h e l o w e r c o n c e n tration range, but tend to become more negative with increasing concentration in t h e u p p e r p o r t i o n o f t h e c u r v e . S i m i l a r t r e n d s h a v e b e e n r e p o r t e d a n d TABLE 1 Polarographie d a t a for the r e d u c t i o n of various a m o u n t s of N - n i t r o s o p a r e p t i d e in 0.5 M acetate buffer, pH 4.75 Conc. (ug rnl -~ )
id (vA)
--E 1/2 (mV)
Slope ( u A / u g m l -~)
300 200 100 40 20 10 5 1 0.5 0.3 0.1
149.0 105.9 62.0 30.0 15.9 8.03 4.00 0.788 0.390 0.225 0.078
0.92 0.90 0.88 0.85 0.84 0.83 0.83 0.83 0.83 0.83 0.83
0.50 0.50 0.62 0.75 0.80 0.80 0.80 0.79 0.78 0.75 0.78
62
generally, in a well-buffered medium, are ascribed to specific adsorption of the reactant and/or electrode product [9] _ The effects of temperature and pH on the reduction of N-nitrosopareptide were studied. The dependence of the differential pulse current on the temperature in the range 20-40°C was 0.6% per degree with the greatest change being 1.0% per degree between 20 ard 25°C. The E!, values were shifted to more negative potentials with increasing pH between 1.4 and 5.2, which is in general accord with results on other N-nitrosamines and supports the mechanism of reduction of the protonated species in acidic solution. The reversibility
of the reduction
was assessed by the scan reversal
technique [lo]. Accordingly, in the pulse mode reversible electroreductions produce an anodic wave equal in height to the cathodic wave, whereas totally irreversible electroreductions yield an anodic wave only about one-seventh &he height of the cathodic wave. The value of the cathodic to anodic wave height ratio for iv-nitrosopareptide was 5.2:i suggesting an irreversibie eiectrode process. Classical logarithmic analysis, where values of log (& -T)/i are plotted vs_ values of E, gave n = l-06, which is in good agreement with values of n obtained
for the irreversible
reduction
of N-nitrosoproline
[ 111.
Dependence of the limiting current on the mercury head pressure was studied in the d-c. mode with a drop time of 2 s. For diffusion-controlled waves the height of the wave is approximately proportional to h213 when a mechanically-controlled drop time is used [ 11 ] _ A bivariate curve-fitting nrnmsm r- -=-----
\Va
I_lSed
to
~aiar!atP
the
nnwer r-. --
of
h- I snd ---
values
of
0-72
and
0-68
were found for concentrations of 20 and 300 r_cg ml-‘, respectively. These results suggest that the limiting currents are diffusion-controlled. Figure 1 shows typical differential pulse polarograms of N-nitrosopareptide at concentrations between 0.1 and 1.0 r.lgml- ‘_ For a drop time of 1 s, a current range of 1 PA, and a modulation amplitude of -100 mV, the detection limit was 0.05-0.1 r.lgml-’ or 1.5-3.0 X lo-’ M.
Derivative
formation
and characterization
,Secondary amines react with nitrous acid, which can be generated in the presence of the amine by the action of mineral acids on sodium nitrite. The product is the polarographically reducible nitrosamine. Pareptide reacted with nitrous acid by virtue of the secondary amine in the proline segment of the tripeptide chain. Nitrosation was complete in 1 h at room temperature in an acetic acid medium, but failed to give a measurable product when the reaction was conducted in dilute mineral acids, e.g., HzS04, HCI and HClO+ Figure 2 illustrates the dependence of the rate of nitrosation on the concentration of sodium nitrite and on the reaction time. The concentration of acetic acid played a less important role in the nitrosation reaction, the same mt.p hpintx nht;tind nt nf L, 5 .7 fl and 25% _...“I YWY’~ _1”--1.-_I rnnrpnt.mt.innc ~~~.YII.Y~L-“~VIIY VA ..v -au .d&.“. Nitrite is reduced at nearly the same potential as N-nitrosopareptide and must be completely removed before the polarographic measurement. The addition of ammonium sulfamate efficiently destroys the excess of nitrite from the reaction.
63
QIOLA
Fig. 1. Differential pulse polarograms of N-nitrosopareptide in acetate buffer pH 4.75; (a) 1.0 pg ml-‘, (b) 0.5 rg ml-‘, (c) 0.3 pg ml-‘. (d) 0.1 pg ml-‘, and (e) supporting electrolyte.
NoNO 8 IO --..__.____L_._.
CON.
(%I
4 2 L__-_.L__-A__._...-
6
\. i
li’
0
0
!’ I5
30
45
60
90 Tune
‘-_&--A
(mm)
Fig. 2. Influence of the concentration of sodium nitrite and the reaction time on the nitrosation of pareptide at room temperature.
The ix. spectra of N-nitrosoproline and other IV-nitroso amino acids contain a medium band at 1430-1450 cm-’ which is attributed to the N-nitroso group [ 81. This region of the i.r. spectrum of ZV-nitrosopareptide was
64 obscured by other strong absorptions; the spectrum does, however, rule out the occurrence of three o t h e r possible reactions. Reaction of the primary amide with nitrous acid can give the corresponding carboxylic acid while the secondary amide could nitrosate. No bands for carboxylic acid or secondary nitrosamide were present, consequently the primary and secondary amide groups were left intact. Formation of an N-nitro derivative was also ruled out, because i.r. bands for N-nitro were absent. The n.m.r, spectrum of N-nitrosopareptide showed the disappearance of the resonance from the amine proton on the proline moiety and the appearance of a resonance attributable to the methylene protons adjacent to the proline nitrogen. Comparison of the mass spectra of N-nitrosopareptide and pareptide showed ions at m/e 328 and m/e 299, respectively. The origins of these ions and additional mass spectral data are listed in Table 2. Analysis of N-nitrosopareptide by u.v. spectrophotometry showed absorption maxima at 343 and 227 nm. Similar absorptions were observed for N-nitrosoproline. The parent amines in both cases have only single maxima at 262 nm (see Table 3). Other nitroso compounds have similar absorption characteristics [8, 12, 13] while N-nitro compounds do not have the absorption maximum at 340--345 nm [14]. The combination of spectroscopic and polarographic data strongly suggests a derivative containing a single nitroso group bonded to the secondary amine in proline. The nitrosation reaction is indicated by .
o
.
o
_,N~,CH:~C_HH_CH2_C_NH H
2 + H o M O 2 + HOAr
1 hr. ~ lit ,~
CH3 CIH2 C--CH 3 I
CH 3
o o II II - - N ~ C H - - C - - N H - - C H 2 - - C - N H 2 .It. NoO.4~: + H20 !
NI It O
I
CH3 CIH2 C-CH 3 I CH3
Analytical application • Analysis o f pharmaceutical dosage forms can often be complicated by the presence of surfactants that distort, suppress, or completely inhibit a polarographic wave. When the technique of standard addition does not successfully circumvent this Problem. a oreliminary clean-up ~ton , ~ . h,~ ,~m,,1. . . . ,~ ~,, remove the surfactant or isolate the c o m p o u n d of interest before the polarographic measurement. Another approach which has proven successful is dilution of the sample solution to concentrations at or below the low pg ml-' range. This approach simplifies sample preparation and minimizes analysis
66
was obtained from an examination of the Ellz values for pareptide and some of its possible decomposition products after nitrosation by the recommended procedure_ Thus prolylleucine showed a wave at a half-wave potential of -0.90 V and proline a wave at -1_08 V, but no responses were obtained for Ieucylglycine, leucine, methylleucine, glycine or glycinamide at concentrations of 10 pg ml-‘_ These results also support the conclusion that N-nitrosopareptide contains a single nitroso group.
The authors express their appreciation
to C. Orzech and R. Daley for
valuable discussions, to G. &hilling for providing mass spectroscopic data, and to R. Pickering for providing h.p.1.c. data. REFERENCES 1 A. Failli, K. Sestanj, H. U_ immer and M. GStz, Arzneim.-Forsch. Drug Res, 27 (1977) 2286. 2 K. Voith, Arzneim.-Forsch. Drug Res., 27 (1977) 2290. 3 T_ A Pugsley and W. Lippman, Arzneim.-Forsch. Drug Res., 27 (1977) 2293. 4 H. Lund, Acta Chem. Stand., 11 (1957) 990. 5 W. F. Smyth, P. Watkiss, J_ S. Burmicz and H. 0. Hanley, Anal. Chim. Acta, 78 (1975) 81. 6 H_ Burghardt, H. Jager and M. von Stackelberg, J. Electroanal. Chem., 17 (1968) 191. 7 R. Vilvaia and J. Halmekoski. Farm. Aikak, 84 (1975) 171. 8 W_ Lijinsky. L_ Keefer and J. Loo, Tetrahedron, 26 (1970) 5137. 9 G. Pezzatini and R. Guidelli, J. Chem. Sot. Faraday Trans 1, 69 (1973) 794. 10 K_ B. Oldham and E_ P_ Parry, AnaL Chem., 42 (1970) 229. I1 K. Hasebe and J_ Osteryoung, Anal. Chem.. 4’7 (1975) 2412. 12 R_ Zahradnik, E. Svatek and M. Chvapil, Collect. Czech. Chem. Commun., 24 (1959) 347. 13 R. N. Hesgeldine and J. Jandev, J. Chem. Sot, (1954) 961. 14 R_ N. Jones and G_ D. Thorn, Can_ J. Res. 276 (1949) 828.
15 Unpublished
in-house procedure.
DIFFERENTIAL PULSE POLAROGRAPHIC DETERMINATION OF THE N - N I T R O S O D E R I V A T I V E O F A T R I P E P T I D E IN P H A R M A C E U T I C A L DOSAGE FORMS
D. G. P R U E * , F. Q. G E M M I L L J R . , a n d R. N. J O H N S O N
A y e r s t Laboratories Inc., 64 ~iaple St., R o u s e s Point, N Y 1 2 9 7 9 (U.S.A.) (Received 18th September 1978)
SUMMARY A m e t h o d is d e s c r i b e d f o r d e t e r m i n i n g a t r i p e p t i d e ( p a r e p t i d e ) i n t a b l e t s a n d c a p s u l e s , w h i c h is b a s e d o n t h e p u l s e p o l a r o g r a p h i c m e a s u r e m e n t o f its N - n i t r o s o d e r i v a t i v e . T h e e l e c t r o c h e m i c a l b e h a v i o r o f t h e d e r i v a t i v e w a s f o u n d t o b e s i m i l a r t o t h a t o f o t h e r Nn i t r o s a m i n e s with regard to the effects o f t e m p e r a t u r e , pH, a n d m e r c u r y pressure. T h e r e d u c t i o n p r o c e s s is i r r e v e r s i b l e a n d c o m p l i c a t e d b y a d s o r p t i o n . O p t i m u m c o n d i t i o n s for p r e p a r a t i o n o f t h e N - n i t r o s o d e r i v a t i v e , w h i c h a p p e a r t o b e a t y p i c a l , are p r e s e n t e d . S p e c t r o s c o p i c d a t a f r o m i.r., m.s., n . m . r , a n d u.v. a n a l y s e s s u g g e s t t h a t t h e d e r i v a t i v e d i f f e r S ' f r o m t h e p a r e n t c o m p o u n d o n l y i n t h a t it h a s a single N - n i t r o s o g r o u p o n t h e p r o l i n e s e g m e n t o f t h e t r i p e p t i d e c h a i n . R e p l i c a t e a s s a y s o n t a b l e t s c o n t a i n i n g 2 m g o f p a r e p t i d e gave excellent precision with a coefficient of variation of 0.37%. C o m p a r i s o n of results o b t a i n e d on capsules containing 2 mg of pareptide and stored at high temperatures showed good c o r r e l a t i o n b e t w e e n t h e p o l a r o g r a p h i c a n d a n h.p.l.c, m e t h o d . R e s u l t s o b t a i n e d w i t h a n u m b e r o f p o s s i b l e d e c o m p o s i t i o n p r o d u c t s i n d i c a t e t h a t t h e p o l a r o g r a p h i c m e t h o d is specific for pareptide.
P a r e p t i d e , L - p r o l y l - N - m e t h y l - D - l e u c y l g l y c i n a m i d e s u l f a t e , has b e e n s y n t h e sized b y a f o u r - c o m p o n e n t c o n d e n s a t i o n p r o c e d u r e [ 1 ] , a n d has b e e n s h o w n t o h a v e p o t e n t i a l clinical a p p l i c a b i l i t y w i t h r e g a r d t o t h e t r e a t m e n t o f P a r k i n s o n ' s disease a n d d e p r e s s i o n [2, 3 ] . P a r e p t i d e itself d o e s n o t possess e l e c t r o c h e m i c a l a c t i v i t y , b u t it c a n b e c o n v e r t e d b y r e a c t i o n w i t h n i t r i t e t o a p o l a r o g r a p h i c a l l y r e d u c i b l e N - n i t r o s o derivative. T h e r e d u c t i o n o f N - n i t r o s a m i n e s at t h e d r o p p i n g m e r c u r y e l e c t r o d e h a s b e e n i n v e s t i g a t e d a n d is g e n e r a l l y w e l l u n d e r s t o o d . In acidic s o l u t i o n , N - n i t r o s a m i n e s u n d e r g o a n irreversible 4 - e l e c t r o n r e d u c t i o n t o t h e c o r r e s p o n d i n g h y d r a z i n e , a n d in basic s o l u t i o n s u n d e r g o a n irreversible 2 - e l e c t r o n r e d u c t i o n t o n i t r o u s o x i d e a n d t h e p r e c u r s o r a m i n e [4, 5 ] . T h e o b j e c t o f this w o r k was t o d e v e l o p a n a n a l y t i c a l m e t h o d w h i c h was sensitive a n d specific f o r p a r e p t i d e in t h e p r e s e n c e o f possible d e c o m p o s i t i o n p r o d u c t s , a n d s u i t a b l e f o r its d e t e r m i n a t i o n in t a b l e t s a n d capsules. T h e m e t h o d p r e s e n t e d h e r e is b a s e d o n a n i t r o s a t i o n r e a c t i o n c a r r i e d o u t u n d e r m i l d c o n d i t i o n s , a n d s u b s e q u e n t d e t e c t i o n o f t h e derivative b y p o l a r o g r a p h y . This a p p r o a c h has b e e n u s e d in s u c c e s s f u l a n a l y s e s f o r o t h e r p h a r m a c e u t i c a l l y
60
important compounds which have a secondary amine functional group, e.g., ephedrine [6] and adrenaline, methylamphetamine and clorprenaline [7]. The electrochemical behavior of IV-nitrosopareptide, conditions for its formation, and characterization of the derivative are also discussed. EXPERIMENTAL Instrumentation
All polarograms were obtained with a Princeton Applied Research polarographic analyzer and recorder (Model 171) equipped with a mechanical drop knocker (Model 172). A Beckman fiber-junction saturated calomel electrode (SCE) was used as the reference electrode and a platinum wire was employed as the auxiliary electrode. The dropping mercury electrode (DME) had the following characteristics (open circuit) in acetate buffer, pH 4.75, m = 5.08 X 1o-4 g s-‘. The following instrumentation was used for characterization of N-nitrosopareptide: Cat-y U.V. spectrcphotometer model 14, Beckman i.r. spectrophotometer model 12, Varian n.m.r. spectrometer model EM-360, and LKB mass spectrometer model 9000.
Pareptide was synthesized [ 1] at Ayerst Research Laboratories, Montreal, Quebec, Canada. For characterization studies, IV-nitrosopareptide solutions were prepared by dissolving 2 g of pareptide in acetic acid 1 + l), then adding 10 ml of 10% sodium nitrite solution and allowing the mixture to stand overnight at room temperature_ The reaction mixture was evaporated to dryness and extracted with acetone. The acetone was removed under vacuum and the resulting oil dissolved in 10 ml of distilled water. The solution was lyophilized to give an amorphous solid. iv-nitroso-L-(--)-proiine was prepared by the method of Lijinsky et ai. [S] . Colorless crystals were obtained (m-p. 104--105°C; [a]g = -187.4”; C = 0.29 in water). All other chemicals were of laboratory-reagent grade.
Analytical
procedure
for tablets and capsules
Weigh out 5, 10 and 15 mg of pareptide reference standard and quantitatively transfer to separate 100-ml volumetric flasks. Dissolve and dilute to volume with acetic acid (I + 19). Pipet 20.0 ml of each standard into separate 25-ml volumetric flasks, add 2.0 ml of aqueous sodium nitrite solution (70 mg ml-‘) and dilute to volume with water. Mix and leave to stand for 60 min. Pipet 5.0 ml of each reaction mixture into separate 50-ml volumetric flasks containing 5-O ml of 2 M ammonium sulfamate solution_ Swirl, leave for 5 min, and dilute each solution to volume with pH 4.75 acetate buffer (0.5 M sodium acetate and 0.5 M acetic acid). Transfer a quantity of standard solution to the polarographic cell and deaerate for 5 min Calibration_
61 with nitrogen. Insert the electrodes and scan from --0.5 V to --1.2 V at 5 mV s -~, w i t h a d r o p t i m e o f 1 s, a c u r r e n t r a n g e o f 2 0 ~ A a n d a p u l s e a m p l i t u d e o f 100 mY. Measure the peak current at --0.83 Y for each standaxd and construct a calibration curve in the conventional manner. Sample preparation. W e i g h o u t t h e e q u i v a l e n t o f 1 0 m g o f p a r e p t i d e f r o m a tablet or capsule powder pool and quantitatively transfer to a 250-ml e x t r a c t i o n b o t t l e . A d d 1 0 0 . 0 m l o f a c e t i c a c i d (1 ÷ 1 9 ) a n d s h a k e o n a m e c h a n i c a l s h a k e r f o r 6 0 rain. C e n t r i f u g e t h e m i x t u r e a n d p i p e t 2 0 . 0 m l o f t h e c l e a r s u p e r n a t a n t l i q u i d i n t o a 2 5 - m i v o l u m e t r i c flask. P r o c e e d w i t h t h e n i t r o s a t i o n r e a c t i o n as d e s c r i b e d a b o v e . C a l c u l a t e t h e s a m p l e s t r e n g t h f r o m t h e e q u a t i o n : m g / t a b l e t ( o r c a p s u l e ) = U × (Wa/Ws}, w h e r e U = m g o f p a r e p t i d e c o r r e s p o n d i n g t o t h e m e a s u r e d p e a k c u r r e n t , Wa = a v e r a g e t a b l e t o r c a p s u l e w e i g h t (rag), a n d Ws = s a m p l e w e i g h t ( m g ) . It should be noted that N-nitrosamines may have carcinogenic properties, so that due precautions should be taken, RESULTS AND DISCUSSION
Polarographic study T h e N - n i t r o s o d e r i v a t i v e o f p a r e p t i d e gives a single w e l l - d e f i n e d p o l a r o g r a p h i c w a v e in p H 4 . 7 5 a c e t a t e b u f f e r w i t h a p e a k p o t e n t i a l o f - - 0 . 8 3 V vs. S C E . T h e d i f f e r e n t i a l p u l s e c u r r e n t i n c r e a s e s l i n e a r l y w i t h c o n c e n t r a t i o n in t h e r a n g e 0 . t ~ 4 0 ug m l - ' ( T a b l e 1). A t c o n c e n t r a t i o n s a b o v e 4 0 u g m l -~, t h e r e is a c h a n g e i n t h e s l o p e o f t h e c u r v e a n d a s e c o n d l i n e a r r a n g e is o b s e r v e d . T h e E ~/2 v a l u e s r e m a i n e s s e n t i a l l y u n c h a n g e d in t h e l o w e r c o n c e n tration range, but tend to become more negative with increasing concentration in t h e u p p e r p o r t i o n o f t h e c u r v e . S i m i l a r t r e n d s h a v e b e e n r e p o r t e d a n d TABLE 1 Polarographie d a t a for the r e d u c t i o n of various a m o u n t s of N - n i t r o s o p a r e p t i d e in 0.5 M acetate buffer, pH 4.75 Conc. (ug rnl -~ )
id (vA)
--E 1/2 (mV)
Slope ( u A / u g m l -~)
300 200 100 40 20 10 5 1 0.5 0.3 0.1
149.0 105.9 62.0 30.0 15.9 8.03 4.00 0.788 0.390 0.225 0.078
0.92 0.90 0.88 0.85 0.84 0.83 0.83 0.83 0.83 0.83 0.83
0.50 0.50 0.62 0.75 0.80 0.80 0.80 0.79 0.78 0.75 0.78
62
generally, in a well-buffered medium, are ascribed to specific adsorption of the reactant and/or electrode product [9] _ The effects of temperature and pH on the reduction of N-nitrosopareptide were studied. The dependence of the differential pulse current on the temperature in the range 20-40°C was 0.6% per degree with the greatest change being 1.0% per degree between 20 ard 25°C. The E!, values were shifted to more negative potentials with increasing pH between 1.4 and 5.2, which is in general accord with results on other N-nitrosamines and supports the mechanism of reduction of the protonated species in acidic solution. The reversibility
of the reduction
was assessed by the scan reversal
technique [lo]. Accordingly, in the pulse mode reversible electroreductions produce an anodic wave equal in height to the cathodic wave, whereas totally irreversible electroreductions yield an anodic wave only about one-seventh &he height of the cathodic wave. The value of the cathodic to anodic wave height ratio for iv-nitrosopareptide was 5.2:i suggesting an irreversibie eiectrode process. Classical logarithmic analysis, where values of log (& -T)/i are plotted vs_ values of E, gave n = l-06, which is in good agreement with values of n obtained
for the irreversible
reduction
of N-nitrosoproline
[ 111.
Dependence of the limiting current on the mercury head pressure was studied in the d-c. mode with a drop time of 2 s. For diffusion-controlled waves the height of the wave is approximately proportional to h213 when a mechanically-controlled drop time is used [ 11 ] _ A bivariate curve-fitting nrnmsm r- -=-----
\Va
I_lSed
to
~aiar!atP
the
nnwer r-. --
of
h- I snd ---
values
of
0-72
and
0-68
were found for concentrations of 20 and 300 r_cg ml-‘, respectively. These results suggest that the limiting currents are diffusion-controlled. Figure 1 shows typical differential pulse polarograms of N-nitrosopareptide at concentrations between 0.1 and 1.0 r.lgml- ‘_ For a drop time of 1 s, a current range of 1 PA, and a modulation amplitude of -100 mV, the detection limit was 0.05-0.1 r.lgml-’ or 1.5-3.0 X lo-’ M.
Derivative
formation
and characterization
,Secondary amines react with nitrous acid, which can be generated in the presence of the amine by the action of mineral acids on sodium nitrite. The product is the polarographically reducible nitrosamine. Pareptide reacted with nitrous acid by virtue of the secondary amine in the proline segment of the tripeptide chain. Nitrosation was complete in 1 h at room temperature in an acetic acid medium, but failed to give a measurable product when the reaction was conducted in dilute mineral acids, e.g., HzS04, HCI and HClO+ Figure 2 illustrates the dependence of the rate of nitrosation on the concentration of sodium nitrite and on the reaction time. The concentration of acetic acid played a less important role in the nitrosation reaction, the same mt.p hpintx nht;tind nt nf L, 5 .7 fl and 25% _...“I YWY’~ _1”--1.-_I rnnrpnt.mt.innc ~~~.YII.Y~L-“~VIIY VA ..v -au .d&.“. Nitrite is reduced at nearly the same potential as N-nitrosopareptide and must be completely removed before the polarographic measurement. The addition of ammonium sulfamate efficiently destroys the excess of nitrite from the reaction.
63
QIOLA
Fig. 1. Differential pulse polarograms of N-nitrosopareptide in acetate buffer pH 4.75; (a) 1.0 pg ml-‘, (b) 0.5 rg ml-‘, (c) 0.3 pg ml-‘. (d) 0.1 pg ml-‘, and (e) supporting electrolyte.
NoNO 8 IO --..__.____L_._.
CON.
(%I
4 2 L__-_.L__-A__._...-
6
\. i
li’
0
0
!’ I5
30
45
60
90 Tune
‘-_&--A
(mm)
Fig. 2. Influence of the concentration of sodium nitrite and the reaction time on the nitrosation of pareptide at room temperature.
The ix. spectra of N-nitrosoproline and other IV-nitroso amino acids contain a medium band at 1430-1450 cm-’ which is attributed to the N-nitroso group [ 81. This region of the i.r. spectrum of ZV-nitrosopareptide was
64 obscured by other strong absorptions; the spectrum does, however, rule out the occurrence of three o t h e r possible reactions. Reaction of the primary amide with nitrous acid can give the corresponding carboxylic acid while the secondary amide could nitrosate. No bands for carboxylic acid or secondary nitrosamide were present, consequently the primary and secondary amide groups were left intact. Formation of an N-nitro derivative was also ruled out, because i.r. bands for N-nitro were absent. The n.m.r, spectrum of N-nitrosopareptide showed the disappearance of the resonance from the amine proton on the proline moiety and the appearance of a resonance attributable to the methylene protons adjacent to the proline nitrogen. Comparison of the mass spectra of N-nitrosopareptide and pareptide showed ions at m/e 328 and m/e 299, respectively. The origins of these ions and additional mass spectral data are listed in Table 2. Analysis of N-nitrosopareptide by u.v. spectrophotometry showed absorption maxima at 343 and 227 nm. Similar absorptions were observed for N-nitrosoproline. The parent amines in both cases have only single maxima at 262 nm (see Table 3). Other nitroso compounds have similar absorption characteristics [8, 12, 13] while N-nitro compounds do not have the absorption maximum at 340--345 nm [14]. The combination of spectroscopic and polarographic data strongly suggests a derivative containing a single nitroso group bonded to the secondary amine in proline. The nitrosation reaction is indicated by .
o
.
o
_,N~,CH:~C_HH_CH2_C_NH H
2 + H o M O 2 + HOAr
1 hr. ~ lit ,~
CH3 CIH2 C--CH 3 I
CH 3
o o II II - - N ~ C H - - C - - N H - - C H 2 - - C - N H 2 .It. NoO.4~: + H20 !
NI It O
I
CH3 CIH2 C-CH 3 I CH3
Analytical application • Analysis o f pharmaceutical dosage forms can often be complicated by the presence of surfactants that distort, suppress, or completely inhibit a polarographic wave. When the technique of standard addition does not successfully circumvent this Problem. a oreliminary clean-up ~ton , ~ . h,~ ,~m,,1. . . . ,~ ~,, remove the surfactant or isolate the c o m p o u n d of interest before the polarographic measurement. Another approach which has proven successful is dilution of the sample solution to concentrations at or below the low pg ml-' range. This approach simplifies sample preparation and minimizes analysis
66
was obtained from an examination of the Ellz values for pareptide and some of its possible decomposition products after nitrosation by the recommended procedure_ Thus prolylleucine showed a wave at a half-wave potential of -0.90 V and proline a wave at -1_08 V, but no responses were obtained for Ieucylglycine, leucine, methylleucine, glycine or glycinamide at concentrations of 10 pg ml-‘_ These results also support the conclusion that N-nitrosopareptide contains a single nitroso group.
The authors express their appreciation
to C. Orzech and R. Daley for
valuable discussions, to G. &hilling for providing mass spectroscopic data, and to R. Pickering for providing h.p.1.c. data. REFERENCES 1 A. Failli, K. Sestanj, H. U_ immer and M. GStz, Arzneim.-Forsch. Drug Res, 27 (1977) 2286. 2 K. Voith, Arzneim.-Forsch. Drug Res., 27 (1977) 2290. 3 T_ A Pugsley and W. Lippman, Arzneim.-Forsch. Drug Res., 27 (1977) 2293. 4 H. Lund, Acta Chem. Stand., 11 (1957) 990. 5 W. F. Smyth, P. Watkiss, J_ S. Burmicz and H. 0. Hanley, Anal. Chim. Acta, 78 (1975) 81. 6 H_ Burghardt, H. Jager and M. von Stackelberg, J. Electroanal. Chem., 17 (1968) 191. 7 R. Vilvaia and J. Halmekoski. Farm. Aikak, 84 (1975) 171. 8 W_ Lijinsky. L_ Keefer and J. Loo, Tetrahedron, 26 (1970) 5137. 9 G. Pezzatini and R. Guidelli, J. Chem. Sot. Faraday Trans 1, 69 (1973) 794. 10 K_ B. Oldham and E_ P_ Parry, AnaL Chem., 42 (1970) 229. I1 K. Hasebe and J_ Osteryoung, Anal. Chem.. 4’7 (1975) 2412. 12 R_ Zahradnik, E. Svatek and M. Chvapil, Collect. Czech. Chem. Commun., 24 (1959) 347. 13 R. N. Hesgeldine and J. Jandev, J. Chem. Sot, (1954) 961. 14 R_ N. Jones and G_ D. Thorn, Can_ J. Res. 276 (1949) 828.
15 Unpublished
in-house procedure.