AValidated HPLC Method for Assay of Morphine Hydrochloride and Hydromorphone Hydrochloride in Pharmaceutical Injections

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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



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



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



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



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

d e t e r m i n a t i o n o f H M H i n v i a ls p r o d u c e d

i n - h o u s e .

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