avalidated hplc method for assay of morphine hydrochloride and hydromorphone hydrochloride in...
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Validated HPLCMethod for ssay of Morphine
Hydrochloride and Hydromorphone
Hydrochloride in Pharmaceutical Injections
2001 ,
53, 35-39
E Smet 1 / G Van der Weken 1/ W R G Baeyens ~/J R Remon 2
1 Laboratory of Drug Quaby Control, Facultyof Pharmaceutical Sciences,Ghent U niversi~y,Harelbekestraat 72, 9000 Ghent, Belgium
2 Laboratory of Pharmaceutical Technology, Faculf,/of Pharmaceutical Sciences,Ghent U niversi~y,Harelbekestraat 72, 9000 Ghent, Belgium
eyWords
Column liquid chromatography
Morphine hydrochloride
Hydromorphone hydrochloride
Pharmaceutical analysis
Validation
Summary
A sensitive and rapid routine HPLC method is proposed for quantitative estimation of mor-
phine hydrochloride and hydromorphone hydrochloride in pharmaceutical dosage forms.
The drugs were chromatographed on a C18 reversed-phase column; the mobile phase was
acetonitrile-water, 35:65 v/v), containing sodium dodecyl sulphate (0.5 , w/v), as ion pair-
ing reagent, and acetic acid (0.4 v/v). Detection was at 230 nm.
The optimized method was validated, and lineari~y r > 0.999), precision, and accuracy were
found to be acceptable within the concentration ranges 86 - 124 I~g mL 1 for morphine hydro-
chloride and 6 0 - 180 I~g mL 1 for hydromorphone hydrochloride.
The method is being used to investigate the stabilif,/of morphine hydrochloride and hydro-
morphone hydrochloride in solutions used for intramuscular njection.
n t roduc t i on
Morphine (F igure 1A), the most impor-
tan t op ium alkalo id , i s used to con t ro l
mo d era t e t o seve re p a i n. Hy d ro mo rp h o n e
(Figur e 1B) is a sem i-synthetic derivative
and valence i somer o f morphine w i th s imi-
lar act ion and uses bu t g reater analgesic
po tency . I t i s p repared by hydrogenat ion
of the 7 ,8 -double bond of morphine and
subsequent ox idat io n of the hydroxyl
group . Sal t s o f morphine are g iven by
man y d i f fe r en t rou t es. Ad mi n i s t r a t i o n b y
mouth i s p referred fo r terminal cancer
pain whereas paren teral rou tes are used
fo r p o s t o p era ti v e p ai n . I n man t h e ma j o r
d e t o x i fi ca t io n p a t h way fo r m o rp h i n e an d
st ructural ly related drugs i s con jugat ion
wi th g lucuron ic acid . The g lucuron ides
accumulate in the k idneys and are ex-
creted in the u r ine. Morphine i s l iab le to
abuse and i t s supply i s therefore s t r ic t ly
cont ro l led . Dependence and to lerance are
no t a p rob lem w hen used leg i t imately in
pat ien ts w i th op io id-sensi t ive pain .
Th e An a l y t i ca l Ab s t r ac t s d a t ab ase
(1982 1999) reveals that gas (GC) an d l i-
q u i d (LC) ch ro m at o g rap h y a r e u su a l ly r e -
co mmen d ed fo r q u an t i t a t i v e d e t e rmi n a-
t i o n o f mo rp h i n e an d h y d ro mo rp h o n e i n
d iverse matr ices . D etect ion i s usual ly per-
formed by mass spect rometry (coupled to
GC) or e lect rochemical ly (coupled to
LC). Only four methods repor t the use o f
LC with ultraviol et (UV) detec tion [1 4].
Of the methods repor ted in the l i teratu re
on ly two have been app l ie d to pharm aceu-
t ical p reparat ions , and bo th use l iqu id
ch ro mat o g rap h y co u p l ed wit h UV-d e t ec -
tion. Menon et al . [1] injected the opiates
on to a 150mm • 4 .6mm , 5 ixm par t ic le ,
Nucleosil Cxs column and used 60:40 (%
v/v)
water-aceton i t r i le con tain ing
1 (v /v)
glacial acetic acid an d 0 .5%
(w/v)
so d i u m
dodecyl su lphate (SDS) as mobi le phase
(1.5 mL min 1); detec tion was at 280 nm.
Venkateshwaran and Stewar t [2 ] used a
300 m m • 3.9 mm , 10 gm particle , Bon da-
pak co lumn wi th 1 :1 phosphate buffer
(pH 6 , 20 mM)-aceton i t r i le as mobi le
phase (1 m Lm in 1), detectin g at 235 nm.
This pape r repor ts a sensi t ive reversed-
p h ase HP LC met h o d fo r th e q u an t i ta t i v e
d e t e rmi n a t i o n o f mo rp h i n e h y d ro ch l o r i d e
(M H) an d h y d ro mo rp h o n e h y d ro ch l o r i d e
(HMH) in so lu t ions used fo r in t ramuscu-
lar in ject ion. The m ethod i s app l icab le to
OH OH OH 0
B
Figure 1. The structures of (A) morphine and
(B) hydromorphone.
Orig inal
0009-5893/00/02 35-05 $ 03.00/0
Ch ro mat o g rap h i a 2001, 53, Jan u ary No. 1/2)
9 2001 Fr iedr . Vieweg & Sohn Ve r lagsgesel lschaf t mb H
35
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routine analysis. It is based on the ion-
pairing properties of SDS with alkaloids
on a narrow-bore RP 18 colum n and UV-
detection at 230 nm. The me thod complies
with validati on requirements in the phar-
maceutical industry.
E x p e r i m e n t a l
R eagents and C h em ica ls
HPLC-grade acetonitrile and SDS were
obtained from Panreac-Quimica (Spain).
Acetic acid was purchased from Acros
(Belgium). MH and HMH were obtained
from Belgopia (Belgium). Deionized
water was used throughout. Other chemi-
cals were of anal ytica l grade.
I ns t r um enta t ion C hr om atogr aph ic
C o n d i ti o n s a n d M o b i l e P h a s e
Chromatography was performed with a
Varian (USA) 9010 SDS pump, a Rheo-
dyne 7125 injec tor with 20-lxL loop, and a
Hewlett-Packard (Germany) series 1050
diode-array detector with 10 mm flow cell
(volume 5 ixL). Integrat ion of c hromato-
grams was performed with Hewlett-Pack-
ard software. Compou nds were separated
on a 125mm • 3m m, 5lxm particle, Li-
Chrospher Ecocart RP-18 column pro-
tected by a 4-4 guard column containing
the same packing (Merck Belgolabo). The
mobile phase was 35:65
v / v )
acetonitrile-
water containing 0.5%
w/v )
SDS and
0.4%
v / v )
acetic acid; the flow-rate was
0.7 mL min 1. Chromatogra phy was per-
formed at 40 ~ The column eluent was
monitored at 230nm, a suitable wave-
length selected from the UV spectra of
MH and HMH in 0.9%
w/v )
sodium
chloride solution.
P r epar a tion o f S tandar d S o lu t ions
o f M H a n d H M H
Separate stock standard solutions of MH
and H MH (600 ixgmL 1) were prepared
threefold in 0.9%
w/v )
sodium chloride
solution. Working standard solutions
were prepared by dilution of the stock
standard solutions with 0.9%
w/v )
so-
dium chloride solution to give solutions
con taining between 60 and 180 ixg mL 1
MH or HMH. Calibration plots of MH
and H MH peak area as a function of con-
centration within this range were estab-
lished with HMH (120 ixgmL 1) as inter-
nal standar d (IS) for MH calibration, and
with MH (120lxgmL 1) as IS for HMH
calibration.
P r epar a tion o f S am ple S o lu t ions
f o r D e t e rm i n a ti o n o f M H a n d H M H
Dilutio ns of intramuscu lar injection solu-
tion from vials containing 100mg MH
and 450 mg NaC1 in 50 mL water (i. e. la-
bel claim 2m gm L 1) were prepared in
0.9%
w/v )
sodium chloride solution down
to a concen tra tion of 120 ixgmL 1, in the
presence of HM H (120 ixgmL 1) as IS.
Dilutions of intramuscular injection
solutions from ampoules containing
10mg HMH, 2mg sodium citrate, and
2 mg citric acid in 1 mL water (i. e. label
claim 10 mg mL 1) were prepared in 0.9%
w/v )
sodium chloride solution down to a
conc ent rat ion of 120 ixgmL 1, in the pre-
sence of MH (120 ixg mL 1) as IS.
Test ing of the Stabi l i ty of M H
i n V i a ls a n d H M H in A m p o u l es
Three batches of MH i ntramu scular injec-
tion solution (label claim 2mgmL 1)
were studied after storage at 25 and 40 ~
for 12 months. Each sample was assayed
to determine the concentration of the ac-
tive ingredient immediately before sto-
rage; furthe r assays to dete rmine the effect
of storage at each temperature were then
performed after 3, 6, 9, and 12 months.
The same method was used to study
the stability of three batches of HMH in-
tramuscular injection solutions (label
claim 10 mgm L 1).
Calculat ions for Determ inat ion
o f M H a n d H M H
Calibration graphs constructed by plot-
ting MH/IS peak-area ratios against MH
concent ration for MH stand ard solutions,
or by plotting HMH/IS peak area ratios
against HMH concentration for HMH
standard solutions, were analysed by
least-squares regression.
R e s u lt s a n d D i s c u s s io n
Prel iminary Exper iments
Because the drugs studied are ionizable
and highly soluble in water, reversed-
phase ion-pair chromatography was the
first choice for this investigation. Method
selection the first step in method devel-
opment was based on previous work on
ion-pai r liquid chromatography of opiates
[1]. Counter ions are added primarily to
reduce interactions of the analytes with
accessible residual silanol groups, which
otherwise would result in tailing, poorly
resolved, and highly retained peaks. The
method then needed to be adapted to
equipment and column (125mm • 3 mm,
5 ixm, LiChrospher RP- 18) that were read-
ily available commercially. On the basis of
our experience in LC mi niatu rizat ion [5
13], a narr ow-bore (3 mm i. d.) rather than
conventional (4.6mm i.d.) column was
chosen, because of advantages including
improved sensitivity and reduction of sol-
vent consumption. This adaptation was
achieved by the method of changing-
one-factor-at-a-time .
The initial mobile phase was 30:70
v /
v) acetonitrile-water containi ng 1%
v / v )
acetic acid and 0.5%
w/v )
SDS. When a
mixture of MH and HMH was injected
MH eluted first at 13.1 min, then HM H at
17.2min. At a flow-rate of 0. 8m Lm in 1
the resolution between the opiates was
4.5. The method was then optimized
further to o btain high resolution and peak
areas with acceptable retentio n times.
The effect of injec tion solvent was first
investigated. Injection in mobil e phase so-
lution resulted in less UV absorption by
MH and HMH, and thus smaller peaks.
Because resolution between MH and
HMH improved slightly when they were
injected in 0.9%
w/v )
sodium chloride so-
lution, rather than water, the former was
chosen as injection solvent. The flow was
set at 0. 7m Lm in 1 (0.6, 0.7, and 0.8mL
min 1 were tested but no differences be-
tween peak areas a nd resol ution were ob-
served). Because reducing the concentra -
tion of acetic acid from 1%
v / v )
to 0.4%
v / v )
resulted in a substantial increase in
resolution with retention times remaining
almost constant this concentration was
used in subsequent work.
Although temperature did not substan-
tially affect resolution, it did, as expected,
affect retention times. When the tempera-
ture was increased from 35 ~ to 45 ~ re-
tention decreased by an average of 15%.
Temperatu re was thus set at 40 ~ The
SDS concent ration was not altered.
Alterati on of the conc entra tion of acet-
onitrile had a substantial effect on reten-
tion a nd resolution. As expected, increas-
ing the acetonitrile content from 30 to
6
Chromat ographia 2001,
53 ,
Januar y (No. 1/2) Original
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36
(v /v)
reduced resolution from 4.5 to
2.9 and also resulted in a favourable re-
duction in retention times (from 13.1 to
4.8 min for MH and from 17.2 to 6.0min
for HMH). For more optimum analysis
the acetonitrile content was set at 35
( v /
v). Further reduction of the acetonitrile
content of the mobile phase was possible,
but because the development of a highly
robust me thod was a primary goal, a mini-
mum resolution of 3 was set. The final
composi tion of the mobile phase was thus
35:65 (
v /v )
acetonitrile-water contain-
ing 0.4
( v / v )
acetic acid and 0.5
(w/v)
SDS.
Finally, because of the intention, some
time in the future, to switch to microbore
HPLC, it was decided to use an IS. With
the purpose of using the same method for
determination of both MH and HMH,
and because these two compounds were
very well separated, HMH was used as IS
for determination of MH, and M H for de-
termination of HMH. Both were found to
be useful molecules for the purpose they
have similar chemical structures, are de-
tectable by the same method (having very
similar UV spectra), and their retention
times are not very different.
M e t h o d V a l i d a ti o n
for the D e te r m ina tion o f M H
a n d H M H i n V i a l s
an d A m poules R espect ive ly
A typical chromatogram obtained from
analysis of a sample from a vial of MH is
illustrated in Figure 2. To show that the
performance of the suggested method
guarantees reliable determination of M H
and HMH, the method was validated in
terms of linearity, precision, accuracy,
specificity, and sample solution stability
under the operating conditions selected.
Specif icity
etermination of M H
The method enables the direct determina-
tion of MH in the presence of the sodium
chloride excipient, because no interference
from the latter was observed. The next
step was to demonstrate that chromato-
graphic resolution could be obtained from
the structurally similar morphine N-oxide
(a degradation product of morphine) and
pseudomorphine molecules. Injection of a
mixture of MH, HMH, and morphine N-
8 o i m U
HM
6 o ~
M
=
t f l
,0 / l I I
i
1 0 0 0
2 0 0 0
3 0 0 0 4 0 0 0
5 0 0 0
6 0 0 0 7 . 0 0 0 a o o o
igure 2. Typical chromatogram obtained after injection of sample solution from an MH vial. The
retention times of MH and HMH are 6.2 and 7.8 min, respectively.
oxide (1 mg mL 1) resulted in the chro-
matogram shown in Figure 3 (pseudo-
morphine, not shown in this chromato-
gram, eluted at 74 min when the flow was
0.85 mL min 1. No peaks interfering with
MH could be detected. When, moreover,
standard solutions of MH ( 25pg mL 1)
were stressed thermally, or under acidic,
basic, o r oxidizing conditions, or by appli-
cation of UV light, for a fixed period of
time, no interfering peaks with retention
times similar to that of MH were obtained
from any of the stressed samples.
etermination of HM H
As expected, the presence of the excipients
sodium citrate and citric acid did not re-
sult in interfering peaks. Because no
breakdown products of HMH have been
reported in the literature, solutions con-
taining HMH were artificially stressed as
described above for MH. No peaks inter-
fering with HM H were observed.
Precision
The repeatability of successive injections
was determined by injecting the same
Table 1. Results from recovery experiments
n = 3 ) .
Level of spike Recovery
R S D
( theoretical content)
Morphine hydrochloride
80 99.3 0.9
100 101.1 0.7
120 100.6 0.9
Hydromorphone hydrochloride
50 99.4 0.9
100 99.8 0.6
150 99.9 0.3
stand ard solutions (100 ) six times for
MH and eight times for HMH. Peak area
R S D
was 0.74 for MH and 0.30 for
HMH. When the same sample solution
was injected six times for MH and eight
times for HMH
R S D
values were 0.54 an d
0.50 , respectively (peak area).
Analytical repeatability for MH was
determined in combination with the accu-
racy study (see below). Recove ry data de-
termined in triplicate at each level (80,
100, and 120 ) are given in Table I. The
R S D of results from replicate analyses
shows that the precision of the method is
acceptable.
Original Chro matog raph ia 2001,
53,
Januar y (No. 1/2) 37
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m U
so~
2 6 0 - <
2 4 0 - ~
220 ~
200~
J
ac~
160
1 4 0 2
120,
i 0 0
6o-]
2 0
1
0 ~
- 4 0 ~ s
- 6 0
1
c
r
T i m e m i n )
9 0 0 2 , 0 0 0 3 . 0 0 0 4 . 0 0 0 5 . 0 0 0 6 . 0 0 0 7 , 0 0 0 8 , 0 0 0
Figure 3. Chromatogram obtained from an artificial mixture of MH, HMH, and morphine N-oxide,
a degradation product of morphine ( lm gm L 1 of each), a=mo rphine N-oxide; b= MH ;
e = HMH.
T a b l e II. Regression equations (linearity study).
Slope Standard error Intercept Standard error
Determination o f MH
Day 1 5.929 0.048 0.002 0.006
Day 2 6.045 0.089 0.017 0.012
Day 3 6.197 0.0870 0.016 0.012
Determination o f HM H
Day 1 11.981 0.138 0.041 0.018
Day2 11.742 0.143 0.025 0.019
Day3 11.310 0.183 0.025 0.030
Analytical precision for HMH was as-
sessed by means of replicate analysis of
eight sample solutions all prepared on the
same day. The RSD (0.77 ) showed tha t
the analytical precision of the method was
acceptable.
ccuracy
The accuracy of the method was deter-
mined by analysis of placebo solutions
spiked with known amounts of MH corre-
spond ing to 80, 100, and 120 of the label
claim (for the MH method) and by spiking
with known amounts o f HM H corre-
spondi ng with 50, 100 and 150 of the la-
bel claim (for determination of HMH).
These experiments were performed on
three different days by the same analyst.
The mean results from replicate analyses
indicate the accuracy of the method. The
results for recovery of HMH and MH
from the specific formulations are shown
in Table I.
Linear i ty
A five-point calibration plot, obtained by
analysis of five standard solutions con-
taini ng 50, 80, 100, 130, and 150 MH
(relative to the label claim) and spiked
with a constant concentration of IS
showed the linear dependence of response
and of the MH/IS peak area ratio on M H
concentration. The overall procedure was
repeated three times on different days. A
similar five-point calibration plot was
constructed for the determination of
HMH. For both compounds the linear
plot (r > 0.999) passed through the origin,
enabling the use of one standard solution
only for routine analysis. The characteris-
tics of the regression equations obtained
for both compoun ds are listed in Table II.
R a n g e
The range for the MH method was set at
80 120 of the label claim for MH
(2mgml 1), because the method was
shown to be precise, accurate and linear
within this region. The range for the
HM H method was 50 150 of the label
claim, again because of its proven preci-
sion, accuracy, and linearity within this
range.
Sam ple So lut ion Stabi l ity
The stability of MH sample solutions was
determined after storage in the dark at
room temperature. Sample preparations
were analysed after 0 and at 48 h and the
data were evaluated to determine the per-
centage change in co ntent since time zero.
Because the percentage changes measur ed
were are within • the sample solu-
tions were considered stable at room tem-
perature for up to 48 h. The stability of a
sample of HMH solution was tested for a
period of 24 h. No significant degrada tion
was observed. These results show that the
validated method for these drugs can be
regarded as a stability-indicating.
ppl icat ion of the Developed
and V a l ida ted M ethod to
the Qua nt i t a t ive Determi na ti on
o f M H in V i al s a n d o f H M H
i n mpoul es
The validated method was successfully
used for the quantitative determination of
MH in injection solutions from vials
(1 mL) cont aining 2 mg mL 1. When a na-
lysis was performed immediately after
batch production the mean MH content
38 Chro matog raph ia 2001,
53
Januar y (No. 1/2) Original
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Table I I I . Amount s (%) r emain ing in v i a l s o f MH and in ampoules o f HMH af t e r s to r age fo r 12 eferences
mon ths a t r oom tempe ra ture (RT) and a t 40 ~
B a t c h T e m p . I m m e d i a t e ly a f t er 3 m o n t h s 6 m o n t h s 9 m o n t h s 1 2 m o n t h s
product ion
Res idua l amount o f MH (%)
98G27 RT 98.6
40 ~
98G29 RT 99.6
40 ~
98H05 RT 99.2
40 ~
Res idua l amount o f HM H (%)
98L04 RT 99.1
40 ~
98L09 RT 100.7
40 ~
RT 98 . 6
40 ~
98L10
101.4 100.7 100.5 100.4
101.2 99.4 97.8 96.9
100.0 100.5 100.2 100.0
98.2 99.2 97.3 97.7
98.8 101.1 100.6 100.6
97.5 99.1 96.9 97.5
95.9 95.5 95.8 97.1
95.1 96.0 95.9 96.7
98.7 100.2 100.3 9 9.2
99.7 99.2 99.6 99.5
98.1 98.9 98.8 98.7
98.3 100.0 99.5 99.6
o f in j e c t i o n s o l u t i o n f r o m d i f f e r e n t
b a t c h e s w a s 9 9 . 1 % o f t h e la b e l c l a i m
( R S D
0 .5 1 % ). W h e n t h e s a m e m e t h o d w a s
u s e d f o r q u a n t i t a t i v e e s t i m a t i o n o f t h e
c o n t e n t o f a m p o u l e s c o n t a i n i n g 1 0 m g
m L 1 H M H t he m e a n H M H c o n te n t o f
s a m p l e s f r o m d i f f e r e n t b a t c h e s w a s 9 9 . 5 %
( R S D
1 . 1%) .
S t u d i e s o f t h e s t a b i l it y o f M H i n v ia l s
a n d H M H i n a m p o u l e s s t o r e d a t 2 5 a n d
4 0 ~ f o r 1 2 m o n t h s w e r e a ls o p e r f o r m e d
b y u s e o f t h e ( s t a b i li t y i n d i c a t in g ) H P L C
m e t h o d . P r e l i m i n a r y r e s u l t s a r e l i s t e d i n
T a b l e I I I ; s t a b i l i t y s t u d i e s a r e s t i l l i n p r o -
gres s .
onclusions
A n i s o c r a t ic r ev e r s e d -p h a s e H P L C m e t h -
o d d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f
M H a n d H M H i n i n j e c ti o n s o l u ti o n s h as
b e e n e v a l u a t e d f o r l i n e a r it y , p r e c i s io n , a c -
c u r a c y , a n d s p e c i f i c i t y . T h e M H p e a k r e -
s p o n s e w a s s h o w n t o b e p r e c i s e , a c c u r a t e ,
a n d l i n e a r o v e r t h e ra n g e 8 0 1 2 0 % o f t h e
l a b e l c l a i m ( 2 m g m L 1 ). S a m p l e s o l u -
t i o n s w e r e s t a b l e f o r u p t o 4 8 h . T h u s t h e
v a l i d a t e d m e t h o d f o r th e a s sa y o f M H
m a y b e r e g a r d e d a s a s t a b i l it y - i n d i c a t in g .
T h e s a m e m e t h o d c a n b e u s e d f o r t h e
d e t e r m i n at i o n o f H M H ( 1 0 m g m L 1) i n
a m p o u l e s , a g a i n b e c a u s e o f i ts p r o v e n l i n -
e a r i t y , p r e c i si o n , a c c u r a c y , a n d s p e c i fi c i ty
w i t h i n t h e r a n g e 5 0 1 5 0 % o f t h e l a b e l
c l a i m . T h e s a m p l e s o l u t i o n w a s s t a b l e f o r
a t l e a s t 2 4 h a n d t h e m e t h o d c o u l d a l s o b e
u s e d a s a s t a b i l i ty - i n d i c a t i n g a s s a y f o r t h e
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Received : Jun 13, 2000
Revi s ed manus cr ip t
received: Sep 8, 2000
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