Development and application of an analytical method for the determination of squalene in...

International Journal of Pharmaceutical Analysis, ISSN: 2051-2740, Vol.39, Issue.1 1210

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Development and Application of an Analytical

Method for the Determination of Morphine and

Clonidine in Vaginal Pessaries Priscilla Capra

Department of Drug Science, University of Pavia, Pavia, Italy

Giorgio. Marrubini Department of Drug Science, University of Pavia, Pavia, Italy

Giorgio Musitelli Department of Drug Science, University of Pavia, Pavia, Italy

Franca Pavanetto Department of Drug Science, University of Pavia, Pavia, Italy

Paola Perugini Department of Drug Science, University of Pavia, Pavia, Italy

Corresponding Author Email: [email protected]

ABSTRACT

A simple isocratic reversed-phase high-performance liquid

chromatographic method was developed for simultaneous

determination of morphine and clonidine in vaginal

pessaries. Chromatographic separation was achieved by a

10 mM aqueous sodium dodecyl sulfate acetonitrile

adjusted to pH 3 with phosphoric acid as mobile phase.

Morphine and clonidine showed an average retention time

of 1.5 and 3.3 minutes, respectively. Specificity, linearity,

precision, range, quantitation and detection limits were

assessed. Calibration curves were linear over the

concentration range of 19-465 µg-mL-1

for morphine and

of 0.12-2.91 µg-mL-1

for clonidine. Method developed

was rapid, simple; it was applied to determine morphine

and clonidine in vaginal pessaries evaluating drug release

profiles in an innovative model vaginal application

conditions. Results showed that both drugs are released at

80% of the initial amount loaded into pessaries within 35

minutes. Morphine is released much faster than clonidine

reaching a steady concentration in less than 15 minutes.

Keywords - Morphine, clonidine, chronic non-cancer

pain, HPLC.

1. INTRODUCTION

Nonopioid analgesics are often coadministered with

morphine to increase efficacy. In particular, clonidine is

frequently added to opioids for long-term intrathecal

delivery in the management of chronic pain patients [1, 2].

The analgesic effect of clonidine via its α2 adrenoreceptor

agonist activity has been well documented in animal and

human studies. Clonidine hydrochloride is an imidazoline-

derivative that acts as an hypotensive agent.

Its mechanism of action appears to be the stimulation of

α2-adrenergic receptors in the CNS, primarily the medulla

oblongata [3]. This results in the inhibition of sympathetic

vasomotor centers. It has also been evaluated for the

control of intense, intractable pain caused by cancer and

other conditions, given epidurally, intrathecally or as an

intraventricular infusion. Clonidine hydrochloride

injection containing 0.15 mg/mL is available in some parts

of the world. Unfortunately, in the United States there is

no parenteral dosage form available commercially; only

oral and transdermal dosage forms, transdermal patches,

are marketed [4]. Moreover clonidine is used in the control

of side effects caused by stimulant medications and leads

to an improvement of the cardiovascular stability

adrenergic and during surgery. Morphine is a potent opiate

analgesic and a psychoactive drug. In clinical practice,

morphine is regarded as the gold standard of analgesics

[5]. The opiates are drugs of choice for short-term

treatment of post-surgical and traumatic pain as well as for

long-term treatment of severe pain in cancer patient.

Particularly for surgical patients, it has been reported that

pain and anxiety may cause major discomfort, increase the

risk for postoperative complications, and even prolong

their hospital residence [6]. Morphine is also a potentially

highly addictive substance; it can cause psychological

dependence and physical dependence as well as tolerance.

According to literature, morphine is the most studied and

most commonly used intravenous drug for Patient-

Controlled Analgesia (PCA); however, large or repeated

doses can induce several side effects prolonged sedation,

nausea, vomiting, apathy, reduced physical activity,

dysphoria, constipation, hypotension and respiratory

depression, which can lead to death. Morphine and

clonidine loaded into vaginal pessaries, were studied as a

new pharmacological association for the treatment of a

characteristic chronic pelvic pain, interstitial cystitis. In

literature it has been already studied the association of

morphine-clonidine in intravenous solution. The goal of

our study was to develop a product which avoids side

effect characteristics of the active product ingredients

(APIs). However, actually, nothing has been proposed yet

regarding their association into formulations such as

vaginal pessaries. For this reasons, the aim of this study

was to develop and apply a simple and fast HPLC method

with UV detection to determine and quantify exactly




morphine and clonidine in vaginal pessaries, and examine

the release profile of the APIs when administered in

vaginal formulations. The dosage of drugs (8 mg for

morphine and 50 μg of clonidine) has been chosen from an

expert team of urologists which occupy of a rare disease,

interstitial cystitis. Several methods have been studied for

determination both morphine and clonidine in biological

fluids and they include high-performance liquid

chromatography mass spectrometry (HPLC-MS) [7], high

performance liquid chromatography tandem mass

spectrometry (HPLC-MS/MS) [8, 9] and gas

chromatography-tandem mass spectrometry (GCMS) [10,

11]. Reversed phase high performance liquid

chromatography (RP-HPLC) with a UV detector is

frequently used for the analysis of morphine and clonidine,

although gas chromatography coupled to mass

spectrometry is the most popular technique applied for

drug of abuse screening and confirmation in biological

fluids [12, 13, 14].

2. MATERIALS AND METHODS

2.1 Materials

Morphine was purchased from S.A.L.A.R.S. S.p.a. (Como,

Italy). Clonidine and sodium dodecyl sulfate were

obtained from Sigma Aldrich (Milan, Italy). Betaglucan

(CM-glucan granulate, SD = 0.85) was acquired from

AVG s.r.l.(Bollate, Milano, Italy), Italy; Gelatine (mesh

size 20, batch 4767 was purchased from LAPI

GELATINE S.p.a. (Empoli, Italy). All other chemicals

used in this experiment were of analytical purity grade and

were purchased from Carlo Erba (Milan, Italy).

Vaginal stimulant fluid (VSF) was set up according to the

following composition: NaCl, KOH, Ca(OH), bovine

serum albumin, lactic acid, acetic acid, glycerol, urea and

glucose.

The experimental use of morphine in the present work is

done in accordance with the requirements of the ministry

of health and regular communication with the local health

authority (ASL of Pavia, Italy).

2.2 Formulation Development

Placebo and loaded pessaries were prepared according to

European Pharmacopeia (8th

edition) [15]. An amount of

gelatin pellets was weighed and hydrated for 12 hours in a

known amount of distilled water (1:1 gelatin-water). Beta-

glucan was hydrate overnight into mixture of water and

glycerin. Subsequently gelatin and beta-glucan phases

were separately transferred in a water bath at 85 °C until

complete fusion and solubilization, respectively. Beta-

glucan solution was added to gelatin under gentle manual

stirring in order to obtain a homogeneous mixture. Finally,

morphine and clonidine were added. The formulation

obtained was sonicated and cast in the moulds. Placebo

pessaries were also prepared by the same procedure

without adding the active principle ingredients. All

batches, placebo and loaded pessaries, were stored at 4°C

until use. Table 1 shows the composition of placebo and

loaded vaginal pessaries.

2.2 Instrumentation and Chromatographic

Conditions

Analyses were performed with an Agilent series 1100

HPLC system equipped with a Rheodyne model G1328A

manual injector with a 20µL stainless steel loop, a model

G1311A quaternary pump, a model G1322A online

degasser, a model G1316A thermostatted column

compartment and a model G1314A variable wavelength

UV detector. Data collection and analyses were performed

using the Agilent Technologies 3D ChemStation software

version Rev. B.04.01 working in a Microsoft Windows XP

operating system environment. Drug separation was

achieved by a column Hypersil - ODS 5.0 µm - (53 X 6.6

mm). The flow rate was 1.0 mL-min-1

. The elution was

isocratic with mobile phase 10 mmol-L-1

sodium dodecyl

sulfate, adjusted to pH 3-4 with phosphoric acid, and

acetonitrile (60:40, v/v).

Table 1 - Composition of placebo and loaded batches.

The column temperature was maintained at 37.0±0.5°C;

the detection was monitored at a wavelength of 214 nm.

At the end of each of the analysis sessions, column

washing was performed at 1 mL-min-1

with a methanol -

water mixture (20:80, v/v) for 15 min, and then with

methanol 100%, for 15 min.

2.3 Standard Solutions and Calibration

Graphs for Chromatographic Measurement

Stock standard solutions of morphine and clonidine were

prepared dissolving them into a pessaries-water mixture

prepared as follows. In a glass vial, beta-glucan placebo

pessaries (3.6 g), 5 ml of filtered water, 8 mg of morphine

powder and 0.05 mg of clonidine (aliquots of 430 µL of a

standard solution of clonidine in 1 mg/ml) were mixed at

37 °C.

The mixture was stirred until a homogenous solution was

obtained. Seven levels of diluted stock standards solutions

were prepared in the concentration range of 465-19 µg-ml-

1 for morphine and 2.91-0.12 µg-ml

-1 for clonidine.

Samples in triplicates were made for each concentration.

Peak areas were plotted against the corresponding

concentrations to obtain the calibration graphs.

Samples Glycerin Gelatin Beta glucan Water Morphine Clonidine

Placebo 50 % 10 % 2 % 38 % - -

Loaded 50 % 10 % 2 % 38 % 8 mg 0.05 mg

https://www.researchgate.net/publication/15123020_Gas_chromatographic-mass_spectrometric_procedures_used_for_the_identification_and_determination_of_morphine_codeine_and_6-monoacetylmorphine?el=1_x_8&enrichId=rgreq-282a6f9ee950d225f85ce94daef46d28-XXX&enrichSource=Y292ZXJQYWdlOzI2NzA0MDExNDtBUzoxNTM5OTY1ODUwODI4ODBAMTQxMzcyNzA1NTg3Ng==




2.4 Method Validation

The method was partially validated considering the ICH

guidelines recommendations [16]. The following

validation parameters were studied: specificity, linearity,

accuracy, precision, limit of detection and quantitation.

2.4.1 Specificity

The method specifity was evaluated by injecting extracted

placebos, placebos fortified with both APIs, and standard

solutions. The assays were performed in order to

demonstrate the absence of interference with the elution of

morphine and clonidine. All chromatograms were

examined to determine if compounds of interest co-eluted

with each other or with any additional excipient peaks.

2.4.2 Linearity and Range

Standard calibration curves were prepared with five

calibrators over a concentration range of 232 µg-ml-1

- 19

µg-ml-1

for morphine and 1.45 µg-ml-1

– 0.12 µg-ml-1

for

clonidine. The data of peak area versus concentration were

treated by linear least square regression analysis after

testing the homoscedasticity of the data. Results were

reported as average of three measurements of three

different samples.

2.4.3 Precision

Precision was assessed in terms of repeatability and

intermediate precision. The repeatability was evaluated

considering one sample at a single concentration, namely

at 93 µg-ml-1

for morphine and 0.58 µg-ml-1

for clonidine,

and six replicate standard samples. The measurements

were performed by the same operator, same

instrumentation and samples of the same batch during one

day of work. Intermediate precision was assessed

considering a single concentration and analyzing six

replicate standard samples on two consecutive days. The

measurements were performed by the same operator on

one HPLC system.

2.4.4 Limits of Detection (LOD) and Limits of

Quantitation (LOQ)

The limit of detection (LOD) and limit of quantitation

(LOQ) for the procedure were assayed by applying the

visual evaluation method on authentic samples prepared at

decreasing concentrations, as requested by the ICH

guidelines [16]. The LODs was assessed by establishing

the minimum level at which the analyte could be reliably

detected. The LOQs were considered as the lowest

concentration of analytes in standards that can be

measured with acceptable accuracy and precision.

2.5 Analytical Method Application

2.5.1 Drug Recovery

Three loaded vaginal pessaries, set up in different days

and stocked at 4°C, were examined. Each sample was

transferred in a glass vial with 5 ml of distilled water (93

μg/ml) and mixed until complete dissolution. Sample

analyses were repeated in triplicate, and the results were

expressed as percent ratio of API amount to the total

weight of the pessaries processed. The assay was

evaluated according to European Pharmacopeia

recommendations [15], considering acceptable values of

APIs recovered from the sample when they are between

95% and 105%.

2.5.2 Dissolution of Vaginal Pessaries

Release profile of loaded vaginal pessaries was also

evaluated by a modified in vitro test apparatus in order to

simulate intravaginal conditions and to discriminate the

formulation release performance [17]. The apparatus

reproduces what happens in vaginal site by transferring the

pessary into a glass vial and continuously exposing it to 2

ml of VSF. A torque movement allows toreproduce the

mechanical stress applied to the pessary formulation when

it is administered into vagina. The experiment was set up

in a water bath at 37 ± 1 °C. All the experiments were

carried out in triplicate. Vaginal pessaries were weighed

and transferred to glass vials and 5 ml of vaginal simulant

fluid (VSF) pH 4.5 were added [18]. The vials were

protected from light since morphine is reported to be

unstable upon exposure to light [15].

At scheduled times (2, 5, 7, 15, 25, and 35 minutes) the

solution was stirred for few seconds in order to guarantee

its homogeneity and an aliquot of the supernatant phase

was withdrawn and analyzed. In order to exclude the

presence of possible interfering substances in the VSF

which could affect the APIs determination, a calibration

curve was built by dissolving placebo formulations spiked

with the target analytes and loaded pessaries in this

medium at 37°C.

3. RESULTS AND DISCUSSION

3.1 Method Development and Validation

The main goal of the development and application of the

present chromatographic method was the separation of

morphine and clonidine from the excipients present in the

formulation. The matrix of vaginal pessary presents one

critical aspect: gelatin and beta-glucan interfere with drug

measurement by direct UV spectrophotometry and

therefore sample pretreatment including extraction and

derivatization is mandatory (data not presented). For this

reason, it was decided to design a direct HPLC-UV

method as simple as possible and determine morphine and

clonidine in both placebo formulation and samples using

an in-vitro model for evaluating the pessaries release

performance. The first critical issue was the choice of an

appropriate medium that allowed complete dissolution of

the pessaries and direct HPLC analysis of the solution

obtained. The solvent ought to be able to dissolve the

matrix, solubilize the APIs and avoid the separation of

excipients that would yield non-homogeneous samples and

thus in the end non-accurate measurements. Therefore,

water was selected and the smallest volume of solvent

necessary to dissolve the pessary, namely 2 ml. The

dissolution of the pessaries was accelerated by heating at

37° C because of the complete melting of gelatin at this

temperature. However, the sample, even for small changes

in temperature tended to re-solidify. Since working at




37°C was considered unacceptably complicated, it was

therefore decided to use a greater volume of water, 5 ml,

because it allowed to dissolve the pessaries at room

temperature (about 25°C). The ion-pair reversed phase

chromatographic mode was selected based on past

experience on basic analytes. The mobile phase, consisting

of 40% acetonitrile and 60% water containing 10mM

sodium dodecyl sulfate initially was brought to pH 3 by

adding acetic acid (0.5%, v/v).

The rationale for this choice is that morphine and

clonidine are both basic compounds, which have pKas of

8.0 and 8.05, respectively. Therefore they are ionized

completely at pH about 3 allowing optimal ion-pairing

with SDS, and selective separations on C8 or C18

columns. However, even if peak symmetry and selectivity

were satisfactory, the method showed poor sensitivity due

to the UV absorption of acetic acid in the eluent.

Therefore, phosphoric acid was selected as mobile phase

acidic modifier because it is transparent to UV light at low

wavelengths thus allowing the detection of the APIs

without producing high background chemical noise.

Figure 1 shows the chromatogram of morphine and

clonidine obtained under the selected conditions. The

peaks of morphine and clonidine are symmetric

(asymmetry factors close to 1), sharp and baseline

separated in the chromatogram. In addition it was possible

to maintain a very short run time and a flexible

chromatographic method (a method in which the retention

of the polar analytes, driven by the aqueous component of

the mobile phase, could be changed within a wide range of

values). To overcome the possible separation of sample

components in the column the analysis temperature was

set up at 37°C, and this proved beneficial also for the

precision of the retention times measurements. Finally,

under the chromatographic conditions selected two

maxima of absorption were observed for morphine and

clonidine at 214 and 225 nm, respectively. The

wavelength which maximizes sensitivity is at 214 nm, and

it showed no interferences with other components of the

formulation.

3.1 Method Validation

The present method was partially validated following the

ICH guidelines for analytical method validation [16].

Figure 1 - Chromatograms of morphine, with a retention time of 1.49 minutes, and clonidine, with a retention time of

3.32 minutes.

3.1.1 Specificity

Injections of the placebo formulation and of the placebo

spiked with M and C were carried out to demonstrate the

absence of interfering compounds in the elution time

frame of the APIs. Figure 2 illustrates the results obtained

and demonstrates that there was no interference from other

materials in the formulation, thus confirming the

specificity of the method.




Figure 2 – Chromatogram of placebo batch.

3.1.2 Linearity and Range

For the construction of calibration curves, calibration

standard solutions were prepared over the concentration

range of 19-232 mg-mL-1

for morphine (n=5) and 0.1-2.9

μg-mL-1

for clonidine (n=7). Calibration curves were built

using the peaks areas of morphine and clonidine versus

their concentration levels.

After testing the data collected for homoscedasticity,

linearity in the range of concentrations tested was proven

by inspection of the residuals plot, from the computation

of the coefficients of the curves together with their

confidence intervals and mean correlation coefficients of

r2 > 0.993 for morphine, and r

2 > 0.999 for clonidine

(Tables 2 and 3).

Table 2 - Calibration curve of morphine. All analyses were

carried out in triplicate.

Nominal concentration

(μg/ml) Curve equation

232 y = (66±2)x + (1454±230)

155 R2 > 0.993

93

39

19

Table 3 – Calibration curve of clonidine. All analyses

were carried out in triplicate.

Nominal concentration

(μg/ml) Curve equation

2.91 y = (1078±3)x + (99±4)

1.94 R2 > 0.999

1.45

0.97

0.58

0.24

0.12

3.1.3 LOD and LOQ

The limit of detection (LOD) and limit of quantitation

(LOQ) were measured on placebo samples. Five

chromatograms of placebo vaginal pessaries were

considered and the difference between the maximum and

minimum value of the baseline noise in the region of

elution of the APIs (D) were estimated. The LODs and

LOQs were assessed according to [16], and confirmed by

injecting placebo formulations spiked at the levels

computed. In particular, LOD and LOQ were respectively

of 0.4μg/ml and 1.3 μg/ml for morphine; and of 0.03

μg/ml and 0.08 μg/ml for clonidine.

3.1.4 Repeatability and Intermediate Precision

Repeatability and intermediate precision were also

estimated. Results were expressed as relative standard

deviation% (RSD%) of the mean assay computed for the

loaded pessaries The precision of the method was

considered acceptable if RSD% was less than 2%. The

method showed repeatability values acceptable for both

APIs. In the case of morphine, the RSD% obtained was

always lower than 0.4%, whereas for clonidine RSD% was

lower than 1%. Intermediate precision was always better

than 2%, and the difference of RSD% was related to the

effect of environmental conditions on the matrix of the

pessaries. Actually the formulation could be affected from

the release or absorption of water from air humidity over

time, and this consequently could modify slightly its

composition and hence the precision of the assay

measurements.

3.2 Drug Recovery

Dosage uniformity of the APIs in the vaginal pessaries

was evaluated according to European Pharmacopoeia [15].

Different batches were examined to obtain statistical

meaningful data. A batch loading of 96.0 ± 0.3% for




morphine, and of 100.00 ± 0.04% for clonidine. However,

morphine showed a greater variability in dosages for the

batches before analysis. Figure 3 shows the percentage

content of batches 1, 2 and 3, analyzed after one, two and

three weeks of storage in a refrigerator at 4°C protected

from light. Batch 0 was one reference batch in which APIs

determination was carried out immediately after

production. Batch 1 and Batch 2, although they had

acceptable drug content percentage, showed an

appreciable variability; whereas in Batch 3, the reduction

of about 9% in the morphine content is remarkable. This

was ascribed to the storage conditions, and in particular to

temperature and humidity, as reported also by previous

reports [19, 20]. Storage conditions could compromise

structural integrity of morphine, or, alternatively, it may

undergo interactions with the mould, that could give rise

to adsorption, affecting analyte quantification.

Figure 3 - Percentage content of batches analyzed at different days of storage.

3.3 Drug Release Study in Vaginal Simulant

Fluid (VSF)

The present method was used to study the release profile

of morphine and clonidine loaded into vaginal pessaries

incubated into vaginal stimulant fluid (VSF). Vaginal

simulant fluid is a complex medium at pH 4.5. Protein and

organic fraction in the fluid contribute to a low pH. Since

the composition of the VSF was suspected to be able to

affect the reliability of the chromatographic analysis of the

APIs, a solubility test in 5ml of VSF was carried out. The

test was designed in order to assess the reliability of the

method results and to confirm the complete dissolution of

the pessaries in model vaginal application conditions. The

application of the method to samples prepared in VSF

showed that retention times of the APIs were unaffected

by the test. The calibration curves of both APIs showed

coefficients of determination always higher than 0.99.

Repeatability and intermediate precision were verified,

confirming the previous data found for RSD% which were

always lower than 2%. Finally, accuracy as expressed by

the recovery percent was of 97 % and 99% for morphine

and clonidine, respectively.

Drug release profiles from vaginal pessaries were studied.

Figure 4 shows release profiles of morphine and clonidine.

Morphine is released to the medium very rapidly and

reaches a steady concentration within 15 min, in

agreement with the diffusion process. Clonidine, instead,

is released in longer times which are estimated around 25

minutes, at complete disaggregation of the pessaries. A

relevant experimental variability was observed for both

analytes. This was related to the sampling which was

carried out unavoidably on non-homogeneous samples.

Such samples are produced by our apparatus on purpose,

because it was requested to reproduce real-life in-vivo

application conditions.

In conclusion, both APIs were fully released by the

pessaries in VSF and model application conditions,

although morphine reaches a steady concentration in less

than 15 min, whereas clonidine is released completely in

less than 35 min.




Figure 4 – Release profiles of morphine and clonidine from vaginal pessary.

4. CONCLUSIONS

A simple isocratic ion-pairing reversed-phase HPLC

method, here proposed, was found to be specific, accurate,

precise, and linear across the analytical range for two

APIs, morphine and clonidine in a vaginal formulation.

Dosage uniformity evaluation showed a loading rate

acceptable and complying with the relations described in

European Pharmacopoeia. Moreover, it was demonstrated

that morphine in the produced formulation is sensitive to

humidity, light and heat upon storage for 1-3 weeks in

refrigerator. Release profiles of analytes from vaginal

pessaries in an in vitro apparatus designed on purpose

demonstrated that both APIs are released to the 80% level

within 35 min. Morphine is released to the vaginal

medium by diffusion in less than 15 min, whereas

Clonidine is released more slowly, most likely because it

is detained by the pessary matrix.

The analytical method described allowed to separate and

quantify the active load in vaginal pessaries, despite the

complex pharmaceutical formulation. The protocol

developed ensured satisfactory estimates of the dose of

clonidine in the formulation, despite its low concentration

relative to the other API, morphine.

5. ACKNOLEGMENTS

The authors thank Mr. Stefano Sacchi for technical

support to this work.

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Development and application of an analytical method for the determination of squalene in...

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Transcript of Development and application of an analytical method for the determination of squalene in...