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Chapter 3 - LEVORPHANOL 46 | P a g e

3.1 DRUG PROFILE

LEVORPHANOL

Levorphanol is an opioid medication used to treat severe pain. It is the

levorotatory stereoisomer of the synthetic morphinan (Dromoran) and a pure

opioid agonist, an orally active morphine-like analgesic. Morphinan is the

parent drug and prototype of a large series of opioid and/or NMDA antagonists

and opioid agonists used in medicine including nalbuphine, but orphanol,

dextromethorphan, and others. Levorphanol labeled with tritium was used in the

research which led to the discovery of opioid receptors in the human nervous

system, including the first study published in 1971. [1] Levorphanol has the same

properties as morphine with respect to the potential for habituation, tolerance,

physical dependence and withdrawal syndrome. It is 4 to 8 times as potent as

morphine and has a longer half-life. Approximately 30 mg of oral morphine is

roughly equianalgesic to 4 mg of oral levorphanol.[2] The levo isomer is the

source of the narcotic properties of the racemic drug Dromoran, but the dextro

isomers are also useful in medicine: in addition to acting on sigma receptors, the

O-methyl derivative of its dextrorotary isomer, dextromethorphan, is a common

NMDA receptor antagonist and pro-drug to dextrorphan.[3] Oral doses of

levorphanol come in 2 mg and 4 mg strengths or subcutaneous injection every 6

to 8 hours. Chemically levorphanol is levo-3-hydroxy-N-methylmorphinan. The

USP nomenclature is 17-methylmorphinan 3-ol tartrate (1:1) (Salt) dihydrate.

The material has 3 asymmetric carbon atoms.

Figure-3A Structure of Levorphanol


Mechanism of action: Like other mu-agonist opioids it is believed to act at

receptors in the peri-ventricular and peri-aqueductal gray matter in both the

brain and spinal cord to alter the transmission and perception of pain. Similar to

many mu-opioid drugs, levorphanol produces euphoria or has a positive effect

on mood in many individuals.

Physicochemical properties of Levorphanol: Levorphanol tartrate is a potent

opioid analgesic with a molecular formula of C17H23NO • C4H6O6 • 2H2O and

molecular weight 443.5. Each mg of Levorphanol tartrate is equivalent to 0.58

mg Levorphanol base. Levorphanol’s chemical name is levo-3-hydroxy-N-

methylmorphinan. The USP nomenclature is 17-methylmorphinan 3-ol tartrate

(1:1) (Salt) dihydrate. The material has 3 asymmetric carbon atoms. It is a white

odorless, crystalline powder. It is sparingly soluble in water, slightly soluble in

alcohol, and is insoluble in chloroform and ether. It melts at 109 degrees with

decomposition.

Clinical Indications: Levorphanol is indicated for the management of moderate

to severe pain or as a preoperative medication where an opioid analgesic is

appropriate. Clinical trials have been reported in the medical literature that

investigated the use of levorphanol as a preoperative medication, as a

postoperative analgesic, and in the management of chronic pain due primarily to

malignancy. In each of these clinical settings Levorphanol has been shown to be

an effective analgesic of the mu-opioid type and similar to morphine,

meperidine, or fentanyl.

Levorphanol has affinity to μ, κ, and δ opioid receptors, but lacks

complete cross-tolerance with morphine. The duration of action is generally

long compared to other comparable analgesics, and varies from 4 hours to as

much as 15 hours. For this reason levorphanol is useful in palliation of chronic

pain and similar conditions. Levorphanol's sigma receptor, SNRI properties

make it even more useful particularly for neuropathic pain. [4]


IUPAC Name : 17-methylmorphinan-3-ol

Formula : C17H23NO

Molecular weight : 257.371 g/mol

Solubility : Methanol

Routes of Administration : oral, intravenous, subcutaneous, intramuscular

The most common side effects persist or become bothersome when using

Levorphanol are Altered mood and thoughts, constipation, dizziness,

drowsiness, flushing, nausea, reaction or pain at the injection site, vomiting.

These severe side effects occur when using Levorphanol: Severe allergic

reactions (rash, hives, difficulty in breathing, and tightness in the chest, swelling

of the mouth, face, lips, or tongue), depression, difficulty in urinating, fainting,

fast, slow, or irregular heartbeat, itching, seizures.

The list of brand names of Levorphanol listed in Table 3.1

Table 3.1 List of Brand Names of Levorphanol

S. No. Brand Name Formulation AvailableStrength Manufacturer

1 LEVO-DROMORAN® TABLET 2mg Valeantpharmaceuticals

2 DROMORAN® TABLET 2mg Roxane laboratories

3.2 LITREATURE SURVEY

Several analytical methods have been reported for the determination of

Levorphanol in pure drug, pharmaceutical dosage forms and in biological

samples using Spectrophotometry, Liquid Chromatography, Electrokinetic

Chromatography and High Performance Thin Layer Chromatography either in

single or in combined forms.

Jason G. Umans et al[5] have described a method for the simultaneous

determination of heroin (3, 6-diacetylmorphine, DAM) and its two active

metabolites 6-acetylmorphine and morphine in blood by high-performance

liquid chromatography using a normal-phase column and a UV detector at 218

nm. The compounds are stabilized in blood by rapid freezing and recovered by


a multistep liquid—liquid extraction. The mobile phase is acetonitrile—

methanol (75:25, v/v) buffered to apparent pH 7 with ammonium hydroxide and

acetic acid. Usingl-α-acetylmethadol as an internal standard, UV detection and a

1-ml biofluid sample, the lower limit of sensitivity is 12.5ng/ml. commonly

used narcotic analgesics including codeine, propoxyphene, meperidine,

methadone and levorphanol do not interfere with the analysis. The method has

been applied to blood samples from humans and rats. Extracts of blood from a

patient who had received an intravenous dose of 14 mg of DAM contained

DAM and both of its active metabolites

John K.Baker et al [6] have described methods for Identification of drugs by

high-pressure liquid chromatography with dual wavelength ultraviolet detection. Using

three solvent-column systems, 101 drugs of forensic interest were characterized

by their high-pressure liquid chromatographic relative retention times and by

the ratio of their absorbances at 254 and 280 nm. Using relative retention times

alone, only 9% of the drugs could be distinguished; while when both the

retention times and absorbance ratios were used, 95% of the drugs could be

distinguished. The compounds were also characterized by comparisons of their

retention times on an adsorption column and a reversed-phase column; however

this pair of discriminators was less useful than the former techniques.

Maria pawula et al [7] have proposed a rapid and sensitive HPLC method for

the determination of codeine, norcodeine and morphine in small volumes of a

biological matrix, using a cyanopropyl column and a combination of

coulometric and UV detection. The compounds were isolated using C18 solid-

phase extraction cartridges prior to quantitative analysis. The limit of detection

was 250µg/ml for morphine and 5ng/ml for both norcodeine and codeine.

Recovery of each compound was greater than 90% and intra- and inter-assay

precision was better than 10%. The method has been used to study the

metabolism of codeine in microsomal incubations

Milan Meloun et al [8] has proposed the mixed dissociation constants of two

drugs—but orphanol and zolpidem at temperatures of (25 and 37) °C were

determined with the use of multi wavelength and multivariate treatments of


spectral data using SPECFIT/32 and SQUAD(84) nonlinear regression. The

factor analysis in the INDICES program predicts the correct number of

components, that is, the number of dissociated and non dissociated forms of the

molecules studied. The thermodynamic dissociation constant pKaT was

estimated by nonlinear regression of {pKa, I} data at (25 and 37) °C: for but

orphanol pKa,1T = 9.46(1) and 8.99(3) and pKa,2

T = 9.64(2) and 9.34(3); for

zolpidem pKa,1T = 6.33(3) and 6.14(1), where the standard deviation in last

significant digits is in parentheses. The proposed procedure involves chemical

model building, calculating the concentration profiles, and fitting the

protonation constants of the chemical model to multi wavelength and

multivariate data measured. If the proposed protonation model represents the

data adequately, the residuals should form a random pattern with a normal

distribution N (0, s2), with the residual mean equal to zero, and the standard

deviation of residuals being near to experimental noise. PALLAS and MARVIN

predict pKa based on the structural formulas of drug compounds in agreement

with the experimental.

3.2.1 Motivation for the method development: The primary purpose of this

research project was to develop and to validate a simple, precise and accurate

HPLC method for determination of Levorphanol in the Bulk and in finished

product. Levorphanol is indicated for the management of moderate to severe

pain or as a preoperative medication where an opioid analgesic is appropriate.

Clearly, it is highly important to accurately measure its concentration alone or

in combination with other compounds. A high speed method was sought to

measure the concentration of this compound within a short span of time. This is

beneficial in any pharmaceutical analysis/clinical environment. The high speed

method will eliminate/reduce any waste or costs that are required with the

preparation of the raw material.

There is no analytical method that has been reported for the determination of

Levorphanol in Bulk and Formulations at the time of commencement of

research work. The other methods reported mainly on the determination of

Levorphanol in plasma and blood samples. Such methods may not be suitable


for regular/routine analysis for Levorphanol in pharmaceutical industry because

of diversity and complexity in sample matrix. The determination of

Levorphanol in a Raw Material sample is yet to be found. In addition, stability-

indicating methods have been able to be found for the Levorphanol in fixed

dosage forms along with other nartcotic drugs. Such simultaneous estimations

are not adopted for single component analysis due to expensive factors.

Complete validation parameters were not able to be found in any of the methods

completed in the past. Studying the stability of a drug and being able to

monitor degradation products aids in the clinical treatments/early product

development and shelf life for the drug. One specific radioimmunoassay (RIA)

procedure has been developed for the determination of the narcotic analgesic,

levorphanol, in plasma using a rabbit antiserum to an albumin conjugate of (-)-

3-hydroxy-N-carboxymethylmorphinan. Such analytical methods based on have

major disadvantage that they are time consuming & cumbersome and laborious

and expensive. Also they are not suitable for regular/routine analysis in

pharmaceutical industry where sample size is more. Hence, by considering all

these factors, the author has made some humble attempts, hoping to fill this gap,

and succeeded in developing analytical methods using HPLC methods.

3.3 EXPERIMENTAL

3.3.1 Instrumentation

The analysis of the drug was carried out on Shimadzu HPLC model (VP series)

containing LC-10AT (VP series) pump, variable wave length programmable

UV/visible detector SPD-10AVP and rheodyne injector (7725i) with 20µl fixed

loop. Chromatographic analysis was performed using Inertsil ODS C-18 column

with 250 x 4.6mm internal diameter and 5µm particle size. Shimadzu electronic

balance (AX-200) was used for weighing.

3.3.2 Chemicals and Solvents

Tablets of combined dosage form were procured from the local market. Other

reagents used like Acetonitrile, Methanol, Ortho phosphoric acid are of HPLC

grade were purchased from E.Merck, Mumbai, India

3.3.3 The Mobile Phase


A mixture of solvents like Acetonitrile, Methanol and 1% orthophosphoric acid

in the ratio of 45:25:30%, v/v was prepared and used as mobile phase.

3.3.4 Standard Solution of the Drug

An accurately weighed sample of 100mg of Levorphanol (working standard)

was transferred into a 100ml volumetric flask gives 1000µg/ml. The solvent

methanol was added and sonicated to dissolve it completely and made up to the

mark with the same solvent. Further 1ml of the above stock solution was

pipetted into a 10ml volumetric flask and diluted up to the mark with methanol

gives 100µg/ml. Further dilutions were made using the same solution.

3.3.5 Sample (tablet) Solution:

Twenty tablets of (Leveodroman® - 2 mg) were weighed, and then powdered. A

sample of the powdered tablets, equivalent to 10 mg of the active ingredient,

was mixed with diluent in 10 ml volumetric flask. The mixture was allowed to

stand for 1 hr with intermittent sonication for complete solubility of the drug,

and then filtered through a 0.45 μm membrane filter, followed by addition of

diluent up 10 ml to obtain a stock solution to get the primary working sample

solution of 1000 µg/mL. The resultant solution was further diluted to get the

100 µg/mL and then filtered through Ultipor membrane sample filter paper

(0.45µ filter).

3.3.6 Calculations in Validation Studies:

Percentage recovery and area ration were calculated using the followingequation:

% recovery = ([Peak Area] sample / [Peak Area] Standard) x 100For a set of “n” replicate measurements, percentage relative standard diviation

was calculated as fallow: % RSD=SD/Average x 100

The detector sensitivity was determined by calculating the signal to noise ratiousing the following equation: Sensitivity = S/N = Signal / Noise

Signal = Amount of detector response to the peak from the middle of the noise

to the summit of the peak.


Noise = Amount of noise resulting from the detector that is taken from a portionof the baseline without any distortions.

3.4 METHOD DEVELOPMENT

Method development consists of selecting the appropriate chromatographic

conditions / factors like detection wave length, selection and optimization of

stationary and mobile phases. Systematic studies on various conditions / factors

have been done for developing a method by studying each parameter by

keeping all other parameters in constant. The following studies were conducted

for this purpose.

3.4.1 Detection Wavelength

The spectra of diluted solutions of the Levorphanol in methanol (at 1ppm and

3ppm concentration) were recorded separately on UV spectrophotometer .UV

absorption spectra revealed that the analyte had absorption maxima between

220 and 230 nm. To obtain the best signals, measurements were performed at

increasing wavelengths from 210 to 250 nm. (at 5nm interval). The largest

signal-to-noise ratio was obtained at 220 nm. PDA-wavelength detection at 228

nm was therefore chosen to obtain good sensitivity for the compound.

3.4.2 Selection of Stationary Phase Column chemistry with respective to the

compound polarity, solvent selectivity (solvent type), solvent strength (volume

fraction of organic solvent(s) in the mobile phase), additive strength, and detection

wavelength, were varied to determine the stationary phase giving the best

separation. Two analytical columns and various mobile phase compositions

were tried in order to reach an acceptable separation as well as a reasonable

chromatographic run time. A cyano-column was highly retentive for the

analytes, and thus resulted in late eluting peaks especially for Levorphanol (tR

24 min). In contrast, the base line chromatographic separation of the target

analyte was accomplished on an octyl-silica column of intermediate

hydrophobicity in 14 min under the described elution conditions.


Review of the literature found a variety of separation columns used in the

analysis of Levorphanol. From among those, the Waters Symmetry C8, 3.5 µm,

4.6 mm x150 mm ID, column (Waters Part No.WAT200630) was chosen for

use initially. However, this column was prone to fouling and also caused

significant back pressure in the HPLC, even when using a 5.0µm Symmetry

guard column ahead of the column. After the initial evaluation of analytical

range, this column was replaced with an Inertsil ODS C-18 column, 5.0 µ, 250

mmx4.6 mm ID. Both columns demonstrated acceptable separation of the

components, but the C18 column was selected for all subsequent analyses on

the HPLC because it was much less prone to fouling and did not cause

exceptional backpressure. Also it produced symmetrical peaks with good

resolution.

3.4.3 Selection of the Mobile Phase

To develop the efficient and reproducible method, different mobile phase

concentrations and rations were employed. Our preliminary trials using various

compositions of mobile phase consisting of water, methanol, Acetonitrile and

different ratios of this solution, did not give good peak shapes, resolutions and

analysis time. Additions of 0.5% o-phosphoric acid to mobile phase at pH 5.2

instead of water improved the peak shape of all investigated compounds as well

as the Pemetrexed disodium. In contrast to gradient mode of RP-HPLC,

isocratic elution needs only one pump, is cost-effective, and can easily be

adopted in most laboratories. Thus, isocratic elution was preferred in this study.

For analysis of compounds, for example, Pemetrexed disodium and its process

related impurities with ionizable basic groups, pH is an important condition in

the separation. Phosphate buffer as pH-modifier for initial preliminary test trials.

Because phosphate buffer may be deposited in the column and chromatograph

under some conditions, it was replaced with o-phosphoric acid was chosen as

pH modifier. Another reason for employing TFA is its compatibility with PDA

detector with less noise. To obtain the best chromatographic separation and

sensitivity in a short time, different mixtures of methanol, and water containing

o-phosphoric acid, and analytical columns with different packing materials,


were systematically investigated. The best separation was achieved with an

Intersil C18 analytical column and Methanol, Water and o-phosphoric acid.

Finally a mixture of Acetonitrile Methanol and Orthophosphoricacid in the ratio

of 45:25:30%, v/v was proved to be the most suitable combination of mobile

phase among all the tested combinations. The chromatographic peak obtained

was better defined and resolved and almost free from tailing.

3.4.4 Flow Rate

A minimum flow rate and minimum run time results the less usage of solvents.

Flow rate of the mobile phase was tested from 0.8 – 1.5 mL/min for optimum

separation and it was found that 1 ml/min flow rate was ideal for the

successful elution of the analyte.

3.4.5 Optimized Chromatographic Conditions and chromatograms

All the Chromatographic conditions derived from above experiments were

shown in the table 2.2. These optimized conditions were followed for the

determination of Levorphanol in bulk samples, tablet formulations. The

chromatograms of standard, blank, tablet sample were shown in figures 2.B,

3.C, 3.D and respectively.

Table 3.2 Optimized Chromatographic Conditions for EstimationLevorphanol

Mobile phaseAcetonitrile Methanol: Orthophosphoric acid

(45:25:30 v/v)Pump mode Isocratic

Mobile phase pH 5.2Diluent Mobile phaseColumn C18 column (250 X 4.6 mm, 5μ)

Column Temp AmbientWavelength 228 nm

Injection Volume 20 μLFlow rate 1 mL/minRun time 10 min

Retention Time 5.1 min


Figure 3B Chromatogram of standard solution

Figure 3C Chromatogram of Blank Solution

Figure 3D Chromatogram of Working Sample Solution


3.5 VALIDATION OF THE PROPOSED METHOD

The proposed method was validated [9-25] as per ICH guidelines [10]. The

parameters studied for validation were specificity, linearity, precision, accuracy,

robustness, system suitability, limit of detection, limit of quantification, and

solution stability.

3.5.1 Specificity

The specificity of the method was assessed by comparing the chromatograms

obtained from drug standards and from placebo solution pre-pared from the

excipients most commonly used in pharmaceutical formulations. Each

Levorphanol tablet (LEVO-DROMORAN®) contains 2 mg levorphanol

tartrate, lactose, corn starch, stearic acid and talc. It was found that there is no

interference due to excipients in the tablet (sample) and also found good

correlation between the retention times of standard and sample. The specificity

results are shown in Table 3.3

Acceptance Criteria:

1. No interferences of the peak of interest in the control or the degraded

sample from the potential degradation products. In other words, the

minimum resolution between Levorphanol peak and the nearest eluting

peak should be NLT 1.50.

2. Peak purity of Levorphanol active ingredient should be NLT 0.99.

Table 3.3: Specificity study Results

Name of the Solution Peak Retention Time in Min’s

Blank No Peak

Levorphanol standard 5.1mins

3.5.2 Linearity and Range

Linearity was performed by preparing standard solutions of Levorphanol at

different concentration levels including working concentration mentioned in

experimental condition i.e. 60ppm. Twenty micro liters of each concentration


was injected in duplicate into the HPLC system. The response was read at

228nm and the corresponding chromatograms were recorded. From these

chromatograms, the mean peak areas were calculated and linearity plots of

concentration over the mean peak areas were constructed individually. The

regressions of the plots were computed by least square regression method.

Linearity results were presented in Table 3.4 and the Calibration plot was

provided in fiigure.3.F

Table 3.4 Linearity Results

Concentration LevelConcentration of

Levorphanol(in ppm)

Mean Peak Area

Level -1 5 39215Level -2 10 82456Level -3 20 158963Level -4 30 234358Level -5 40 283670Level -6 50 362983Level -7 60 427183

Range: 5 ppm to60 ppm

SlopeIntercept

Correlation coefficient

7068.62157020.9986

Figure -3E: Calibration Curve for Levorphanol


3.5.3 Method Precision: Precision of the assay method was determined by

repeatability (intra-day) and intermediate precision (inter-day) using the

triplicate analysis of the QC samples. Repeatability shows the applicability of

the analytical procedure within a laboratory over a short period of time that is

evaluated by assaying the QC samples during the same day. Intermediate

precision is assessed by comparing the assays on different days. Precision is the

degree of repeatability of an analytical method under normal operational

conditions. Precision of the method was performed as Intraday precision and

Inter day precision

3.5.3.1 Intraday Precision

To study the intraday precision, six replicate standard solutions (60ppm) of

Levorphanol were prepared and injected. The percent relative standard

deviation (% RSD) was calculated and it was found to be 0.31, which is well

within the acceptance criteria of not more than 2.0%. The results of system

precision studies are shown in Table 3.5.

Table 3.5 Intraday Precision Results for Levorphanol

SampleConcentration(ppm)

InjectionNo.

PeakAreas

R.S.D(Acceptance

criteria ≤ 2.0%)1 794123

2 799863

3 794785

4 793387

5 792810

Levorphanol 60

6 796589

0.31


3.5.3.2. Inter Day Precision

To study the Inter day precision, six replicate standard solutions (60ppm) of

Levorphanol were injected on third day of sample preparation. The percent

relative standard deviation (% RSD) was calculated and it was found to be

0.32%, which is well within the acceptance criteria of not more than 2.0%.

Results of Inter day system precision studies are shown in Table 3.

Table 3.6 Inter day Precision Results for Levorphanol

Sample Concentration(ppm)

InjectionNo.

PeakAreas

R.S.D(Acceptancecriteria ≤ 2.0%)

1 7821342 7865453 7897324 7846135 786577

Levorphanol 60

6 785643

0.32

3.5.4. Accuracy

The accuracy of the method was determined by standard addition method.

Three accuracy batches containing calibration curve standards and six replicates

of QC standard samples at Three concentration levels were performed in mobile

phase/diluents samples were extracted and analyzed in three different runs

along with one standard zero (blank without IS) (one used to check the

interference contribution at the retention time of analytes) A known amount of

standard drug was added to the fixed amount of pre analyzed tablet solution.

Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at

50%, 100% and 150% level of 60ppm. The solutions were analyzed in triplicate

at each level using the proposed method. The percent recovery and % RSD was

calculated and results are presented in Table 2.7. Satisfactory recoveries ranging

from 98.0 to 101.0% were obtained, indicating that the proposed method was

accurate.


Table 3.7 Accuracy Results for LevorphanolAmount of Levorphanol ( in ppm)Level

Spiked Recovered % Recovery20 19.67 98.9520 19.31 96.1550 %20 20.09 100.1540 40.01 10040 39.57 98.7100%40 40.23 100.360 60.38 100.560 60.12 100.3150%60 60.16 100.4

Mean 99.29

3.5.5. Robustness- A method is robust if it is unaffected by small changes in

operating conditions. The robustness was studied by evaluating the effect of

small but deliberate variations in the chromatographic conditions. The

conditions studied were flow rate (altered by ±0.2mLmin−1), mobile phase

composition (acetonitrile±5%), and buffer pH (altered by ±0.2). These

chromato-graphic variations were evaluated in a system suitability solution with

respect to retention time RT and % assay of drugs. Levorphanol at 60 ppm

concentration was analyzed under these changed experimental conditions. Three

replicate injections were performed with each of the altered chromatographic

condition and the mean peak area was compared against the mean peak area

obtained with the unaltered conditions. Under these variations, the analyte was

adequately resolved and elution pattern remained unchanged. The system

suitability solution maintained a signal-to-noise ratio over 10 in all varied

conditions. It was observed that there were no marked changes in

chromatography and the %assay when compared with unaltered conditions was

with in ±2%, demonstrating that the developed method was robust in nature.

The results of robustness study are shown in Table 3.8

Table 3.8 Robustness of LevorphanolCondition Mean Area % Assay % DifferenceUnaltered 427183 100.0 -

Wave length 230 435632 102.9 2.9Mobile phase:ACN : METHANOL: 446831 103.7 3.7


OPA : 45:25:30pH of mobile phase at 4.9 426765 101.8 1.83.5.6. Ruggedness

To study the Ruggedness, six replicate standard solutions (60ppm) of

Levorphanol were injected by another person to determine the person to person

variation. The percent relative standard deviation (% RSD) was calculated and

it was found to be 0.22%, which is well within the acceptance criteria of not

more than 2.0%. Results of Ruggedness studies are shown in Table 3.9 There

had no significant effect on the retention time and chromatographic response of

the method, indicating that the method was rugged.

Table 3.9: Ruggedness of Levorphanol

Sample Concentration(ppm)

InjectionNo.

Peak Areas R.S.D(Acceptancecriteria ≤ 2.0%)

1 7856232 7845983 7826484 7854125 784781

Levorphanol 60

6 788129

1.63

3.5.7 System Suitability

System suitability was studied under each validation parameter by injecting six

replicates of the standard solution. For the method following limits were

considered as acceptance criteria, Tailing factor ≤ 2, Theoretical plates > 2000

and %RSD for peak area ≤ 2%. The system suitability results are given in Table

3.10

Table 3.10 System Suitability Results for Levorphanol

Parameter TailingFactor

TheoreticalPlates

% RSD for PeakResponse

Specificitystudy

1.27 6471 0.6

Linearity study 0.98 7089 0.7

Precision study 1.07 7247 0.5


3.5.8. Limit of Detection and Limit of Quantification

To determine the Limit of Detection (LOD) sample was dissolved by using

Mobile phase and injected until peak was disappeared. After 0.03ppm dilution

Peak was not clearly observed, based on which 0.15ppm is considered as Limit

of Detection and Limit of Quantification is 0.05 ppm. For establishing LOQ, six

replicates of the analyte at 0.02ppm were prepared and quantified with precision

of 1.0%. The LOD and LOQ of Levorphanol are given in Table 3.11

Table 3.11 Limit of Detection and Limit of Quantification for Levorphanol

Parameter Measured Value

Limit of Quantification 0.05ppm

Limit of Detection 0.15ppm

3.5.9 Analysis of Commercial Formulation:

For assay Levorphanol (LEVO_DROMORAN) a composite of 20 tablets was

prepared by grinding them to a fine, uniform size powder. 10 mg of

Levorphanol was accurately weighted and quantitatively transferred into a 100

ml volumetric flask. Approximately 25 ml mobile phase were added and the

solution was sonicated for 15 min. The flask was filled to volume with mobile

phase, and mixed. After filtration, an amount of the solution was diluted with

mobile phase to a concentration of 60μg/ml. An aliquot of this solution was

injected into HPLC system. Peak area of Levorphanol was measured and

compared against the peak area of the standard solution. The proposed method

was able to estimate Levorphanol in the tablet formulation with an accuracy of

98.96%.

3.6. DISCUSSION ON THE RESULTS

The UV spectra of Levorphanol showed absorption maximum at 228nm

which was selected as the detection wave length in the current assay.

Optimization of mobile phase was performed based on chromatographic


separation, peak shape and peak area obtained. Different mobile phases were

tried but satisfactory separation, well resolved and good symmetrical peaks

were obtained with the mobile phase Acetonitrile: Methanol:

orthophosphoricacid in the ratio of 45:25:30%, v/v. The retention time of

Levorphanol was found to be 5.1 min, which is well separated from the solvent

front and from all other interfering peaks. The number of theoretical plates was

found to be >5000, which indicates efficient performance of the column. The

tailing factor was found to be <1.32, which indicates a relatively good

symmetry of the peak of interest. All these values meet the method system

suitability acceptance criteria and were shown in Table 3.10.

The calibration curve for Levorphanol was obtained by plotting the peak

area versus the concentration of Levorphanol over the range of 5-60ppm, and it

was found to be linear with “r” value > 0.995. The regression equation of

Levorphanol concentration over its peak area was found to be y = 7068.62x

+15702 (r= 0.997), where x is the concentration of Levorphanol (ppm) and y is

the respective peak area. The data of regression analysis of the calibration curve

was shown in table 1. Precision was evaluated using six independent

Levorphanol sample solutions (60ppm) prepared in the Mobile phase.

Percentage relative standard deviation (%RSD) was ( for Intraday-0.31, For

Inter day-0.32) found to be less than 2% for within day and day to day

variations, which proves that method is precise. Standard addition method at

50%, 100% and 150% of 40ppm was carried out to evaluate the Accuracy of the

method for estimatation of Levorphanol. The results showed good recoveries

ranging from 98.0% to 101.0%. The mean recovery data obtained for each level

as well as for all levels combined (Table 3.7) was within 2.0% of the label claim

for the active substance with an RSD of < 2.0%, which satisfied the acceptance

criteria set for the study.

The proposed method has been applied to the assay of commercial tablet

containing Levorphanol. Sample was analyzed for three times after extracting

the drug. The results presented good agreement with the labeled content. Low


values of standard deviation denoted very good repeatability of the

measurement.

The Ruggedness of Levorphanol was evaluated by injecting the six replicates of

standard solution (at 60ppm) to determine the variation of result between person

to person. Percentage relative standard deviation (%RSD) was 0.22% found to

be less than 2% which is in the acceptable range. Typical variations in liquid

chromatography conditions were used to evaluate the robustness of the assay

method. In this study, the chromatographic parameters monitored were retention

time, area, tailing factor and theoretical plates. The robustness acceptance

criteria set in the validation were same as established on system suitability test

described above.

Limit of detection (LOD) is defined as the lowest concentration of analyte that

gives a detectable response. Limit of quantification (LOQ) is defined as the

lowest Concentration that can be quantified reliably with a specified level of

accuracy and Precision. The Limit of detection and Limit of quantification for

Levorphanol was found to be 0.05ppm and 0.15ppm respectively.

Conclusion: This method represents a fast analytical procedure for the

simultaneous quantification of Levorphanol in tablet dosage forms. The sample

preparation is simple, the analysis time is short and the elution is isocratic. The

method is amenable to the analysis of large numbers of samples with excellent

precision and accuracy. Based on the presented data, it is concluded that the

High Performance Liquid Chromatography-UV Detection method for

determination of Levorphanol is Specific, Simple and Sensitive. Linearity,

Precision and Accuracy of the method over the concentration range of 5-60ppm

has successfully met the acceptance criteria. The Stability evaluation performed

on both standard solution and on the sample solution (prepared from tablet) has

demonstrated insignificant degradation of Levorphanol over the specified

storage conditions and duration. The current method can be successfully applied

to the estimation of Levorphanol in bulk and in pharmaceutical dosage forms as

part of Quality control analysis and quantification of Levorphanol.

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