Research Article Vol: 1; Issue: 1 Development and Validation of ...ijpda.com/admin/uploads/kBjcGP.pdf · The injection volume is 20µL. The retention time of Diazepam and Propranolol

Research Article

Vol: 1; Issue: 1

Development and Validation of stability indicating method for

the simultaneous determination of Diazepam and Propranolol

Hydrochloride by RP-HPLC.

A Shanmukha naresh1, T. Santosh Kumar

2, S Satyanarayana

1*

3Pharmaceutical R & D Laboratory, Corpuscle Research Solutions, Visakhapatnam-530003, India.

1Avanti Institute of Pharmaceutical Sciences, Vizianagaram Dist, AP, India

Date Received:

6TH Sep 2013

Date of Accepted:

1st Oct 2013

Date Published:

14 Oct 2013

1

Abstract: A simple, selective, rapid, precise and economical reverse phase high pressure liquid chromatographic method has been

developed for the simultaneous estimation of Diazepam and Propranolol HCl in pharmaceutical Tablet dosage form. The

mobile phase consisted of 65:35 % (v/v) of Methanol & 0.1% v/v ortho phosphoric acid operated on isocratic mode. The

flow rate is 1.0 ml/min. Chromatographic separation of Diazepam and Propranolol HCl was performed on Phenomenox

C18 column (150 X 4.6 mm id, ODS 2, 5µm). The wavelength of detection is 222 nm. The injection volume is 20µL. The

retention time of Diazepam and Propranolol Hcl are 7.1 ± 0.10 minutes and 2.1 ± 0.10 respectively. The run time of

analysis is 8.5minutes. The developed method was validated for parameters such as accuracy, precision, linearity, limit of

detection, limit of quantitation and solution stability. The influence of acid, alkaline, oxidative Stress and photolytic stress

conditions on both the drugs was studied. Results indicated complete degradation in alkaline medium for Diazepam and

Propranolol HCl The proposed method has been successfully used for the estimation in tablet dosage forms.

Keywords: Diazepam, Propranolol HCl, HPLC, anti hypertensive, anti anxiety

Introduction

Diazepam (fig 1a) chemically 7-chloro-1, 3-dihydro-1-

methyl-5-phenyl-2H-1,4-benzodiazepin-2-one. The

Molecular formula and Molecular weight are

C16H13ClN2O, 284.75 g/mol respectively. Diazepam is

a benzodiazepine that binds to a specific subunit on the

GABA receptor at a site that is distinct from the

binding site of the endogenous GABA molecule..

Diazepam appears to act on areas of the limbic system,

thalamus and hypothalamus, inducing anxiolytic

effects. Its actions are due to the enhancement of

GABA activity. Benzodiazepines including diazepam

increases the inhibitory processes in the cerebral cortex[1]. Propranolol HCl (fig 1b) chemically is 2-

Propanol,1-[(1-methylethyl)amino]-3-(1-

naphthalenyloxy)-hydrochloride. Propranolol decreased

cardiac output, inhibition of renin release by the

kidneys , and diminution of tonic sympathetic nerve outflow from vasomotor centers in the brain. Propranolol

may increase oxygen requirements by increasing left

ventricular fiber length, end diastolic pressure, and

systolic ejection period [2-3]. Several HPLC methods

have also been reported for the determination of

Propronolol HCl and Diazepam individually [4-5] and in

combination with other drugs [6-12]. However, there is

no simultaneous method reported for their simultaneous

estimation. Hence we have planned to develop a

validated reversed-phase HPLC method for the

estimation of these drugs in combined dosage form as per ICH guidelines [13-17]. The aim of the present study

was to develop accurate, precise and selective reverse

phase HPLC methods for the simulated analysis of

Diazepam and Propranolol HCl.

Available online at www.ijpda.com

A Shanmukha Naresh et al; Vol: 1, Issue: 1, Page: 1-12

2

Fig-1a: Structure of Diazepam

Fig-1b: Structure of Propronolol HCl

2. EXPERIMENTAL

2.1. Reagents and chemicals

Orthophosphoric acid (AR Grade, Merck ltd), Methanol

(HPLC grade, Merck ltd), Milli-Q water, Diazepam (99.8

% w/w is a gift sample from corpus research solutions)

and Propranolol HCl (99.8% w/w purchased from

(corpus research solutions), glacial acetic Acid (GR

Grade, SD Fine Chem Ltd). All other chemicals are of

the highest grade commercially available unless otherwise specified. DIAZEPAX tablets for evaluation of

the assay content were purchased for a local pharmacy.

2.2. Apparatus and chromatographic conditions

The Chromatographic system consisted of a Shimadzu

Class VP Binary pump LC-10ATvp, SIL-10ADvp Auto

sampler, CTO-10Avp Column Temperature Oven, SPD-

10Avp UV-Visible Detector. All the components of the

system are controlled using SCL-10Avp System

Controller. Data acquisition was done using LC solutions

software. The mobile phase consisted of 65:35% (v/v) of

Methanol and 20mM Orthophosphoric acid (pH adjusted to 3.0 with acetic acid) operated on isocratic mode. The

flow rate is 1.0 ml/min.

Chromatographic determination of Diazepam and

Propranolol HCl was performed on Phenomenex C18

column (150 X 4.6 mm id, ODS 2, 5µm). The

wavelength of detection is 222 nm. The injection volume

is 20µL.

2.3. Preparation of standard solutions, Calibration

Standards & Quality Control Samples

Stock solutions of Diazepam (2mg/mL), Propranolol

HCl (0.5mg/mL) were prepared separately in a

volumetric flask using methanol and labeled

accordingly. Suitable dilutions were then prepared using

50:50 %v/v Methanol & Milli-Q water as Diluent

Solution. A Linear Calibration curve containing 8 non-

zero standards were prepared using Diluent solution in the concentration range of 1.00-50.00 µg/mL for

Propranolol HCl & 2.50-50.00 µg/mL for Diazepam.

The calibration standard sample is then transferred into

the auto sampler for analysis. Samples for Specificity

(Sample with Diazepam alone, sample with Propranolol

HCl alone, Blank Sample and sample containing both

the drugs) were also prepared accordingly. For the

preparation of quality control samples, a separate stock

containing approximately the same concentration of the

Diazepam and Propranolol HCl were prepared and

labeled as quality control stocks. From these stocks,

quality control samples containing Diazepam and Propranolol HCl were prepared at three concentration

levels namely LQC, MQC, and HQC so as to obtain low,

median and high concentration quality control samples.

The performance of the linear calibration curve is then

evaluated using quality control samples.

2.4. Assay

The assay of tablets containing Diazepam and

Propranolol HCl (Brand name: DIAZEPAX) is done

using the procedure given in Indian Pharmacopoeia

under tablets. The active ingredients in each of 10 dosage units is taken by random sampling and analyzed

by the developed method. The tablets are said to be

compliant if the each individual content is 85 – 115 % of

the average content or labeled claim. For the current

assay ten tablets were randomly taken and transferred

separately into 100ml volumetric flasks and dissolved in

20 ml methanol. The solution was then ultra sonicated

for 10min and then made up to volume. Required

amount of solution is then taken and filtered through

0.45µ nylon membrane and diluted with diluent solution

so that the resultant concentrations are within the

calibration range of the developed method. The samples are then analyzed by using the validated method. The

sample is then injected in triplicate.

2.5 Method Validation

2.5.1 System Suitability

A sample containing mixture of Diazepam (at

concentration of 25µg/ml) and Propranolol HCl (at

concentration of 10µg/ml) was used as system suitability



3

sample. System suitability was assessed by six replicate

analyses. A percent coefficient of variation (% CV) less

than 1 % for retention times for the drugs is taken as the

acceptance criterion.

2.5.2 Detection and Quantitation Limits (Sensitivity)

Limits of detection (LOD) and quantification (LOQ)

(Fig-2) were estimated from both linearity calibration

curve method and signal to noise ratio method. The detection limit was defined as the lowest concentration

level resulting in a peak area of three times the baseline

noise. The quantification limit was defined as the lowest

concentration level that provided a peak area with signal

to noise ratio higher than 5, with precision (%CV) and

accuracy with (±) 20%.

Fig-2: Chromatograms shown below indicate limit

of Detection (LOQ) above and Limit of

Quantitation (LOD) below.

2.5.3 Linearity (Calibration Curve)

The calibration curve was constructed with eight non-

zero standards ranging from 1.00 to 20.00 µg/mL for

Propranolol HCl and 2.50–50.00µg/mL for Diazepam.

The linearity was evaluated by linear regression analysis,

which was calculated by least square method. It is

depicted in (Fig- 3).

2.5.4 Accuracy and Precision

Accuracy of assay method was determined for both

intra-day and inter-day variations using triplicate

analysis of the QC samples. Precision of the assay was

determined by repeatability (intra-day) and intermediate

precision (inter-day). Repeatability refers to the use of

the analytical procedure within the laboratory over the

shorter period of the time that was evaluated by assaying

the QC samples during the same day. Intermediate precision was assessed by comparing the assays on

different days (2 days).

2.5.5 Specificity

For demonstration of specificity, 4 samples namely

Blank sample, sample containing Diazepam alone,

sample containing Propranolol HCl alone and sample

containing the mixture of Diazepam and Propranolol

HCl were prepared separately. Specificity of the method

was determined by comparing results of all the samples

(Fig-4). The developed method is said to be specific if

the % interference calculated as peak area (if any) at the

retention time of each of the analytes in the blank sample is less than 20% of peak area at the corresponding

retention times of each of the drugs in the lowest

calibration standard. Sample Specificity is also observed

in the degradation study of the drug. None of the

degraded products must interfere with the quantification

of the drug.

2.5.6 Stability

The stability of the drug is determined by placing the

MQC samples for the short term stability at room

temperature up to 12 hours and then comparing the obtained peak area with that of the similarly prepared

fresh sample. Further, auto-sampler stability for up to 24

hrs was studied and established.

2.5.7 Stress Degradation Studies

For Stress Degradation Analysis, 1 mL aliquots (in

duplicate) of samples containing MQC level

concentration are treated separately with 100 µL of 0.1N

HCl (Acid stress), 0.1N NaOH (Alkaline stress), 5% v/v

Hydrogen Peroxide (Oxidative Stress), for 24 Hrs.

Samples for Photolytic stress are placed in a transparent

glass vial & placed in a UV chamber for 24 Hrs. Samples are then injected for analysis. The results of

analysis are then compared with similarly prepared fresh

samples. The analysis is performed in triplicate.



4

3.0 RESULTS AND DISCUSSION

3.1 Method Development and Validation

The HPLC procedure was optimized with a view to

develop a stability indicating assay method. Functional

group analysis revealed the presence of acidic character

to the molecules. Therefore we evaluated the

chromatographic behavior at different pH values ranging

from pH 3.0 to pH 6.5 using various columns like

Hypersil-BDS-C18, Symmetry C18, Ymc-pack C18, Ymc-pack pro, Spherisorb C18, Phenomenex C18 have

been tried with different buffer salts such ammonium

Formate, ortho phosphoric acid, di-potassium hydrogen

orthophosphate, in combination with acetonitrile,

methanol and tetrahydrofuran. However less tailing and

high theoretical plates are obtained with Phenomenex

column C18 250 X 4.6 cm 5µm column.. The peak

response of Propranolol HCl decreased with increased

composition of Methanol in the mobile phase. Mobile

phase composition consisted of (65:35v/v) of Methanol

and 20mM Orthophosphoric acid (pH adjusted to 3.0 ±

0.1 with glacial acetic acid) on isocratic mode. The flow rate of the method is 1.0 ml/min. Calibration standards

were prepared in diluents solution containing 50:50 %

v/v of Methanol and Milli-Q water. The wavelength of

detection is 222nm. The column temperature is

maintained at 25 OC. At the reported flow rate, peak

shape was excellent; however increasing or decreasing

the flow rate resulted in unacceptable tailing factor and

poor peak shape. Hence 1.0 ml/min was optimized flow

rate decreasing the consumption of the mobile phase,

which in turn proves to be cost effective for long term

routine quality control analysis. To evaluate the feasibility of the experiment under regular lab conditions,

we assessed the stability of Diazepam and Propranolol

HCl under room temperature and under normal light

conditions.

3.2 Method Validation

3.2.1 System Suitability

The % RSD of the peak area for both drugs is within the

acceptable criteria (Table-1). The efficiency of the

column was expressed as the number of theoretical plates

for the six replicate injections was around 21334.5 ±1 20

for Diazepam and 11187.2 ± 65 for Propranolol HCl. The USP tailing factor for Diazepam and Propranolol HCl is

not more than 2.0 while that of Diazepam is 1.072 ±0.04

and Propranolol is 1.517±0.04.

3.2.2 Determination and Quantification Limits

(Sensitivity)

Fig-2 represents the chromatogram of limit of detection

and limit of quantification. The method is found to be

sensitive which can be determined from the data

obtained from the (Table-2).

3.2.3 Linearity

The linearity was demonstrated in triplicate. The results

of the best fit line (y = mx + c) for the triplicate analysis

is given in Table 3. The accuracy of the calibration

standards was evaluated from the back calculated concentrations (Table 4). All the standards were found

to be within the range of 95.97 to 108.17 % for

Diazepam and 94.00-108.78 % for Propranolol.

3.2.4 Accuracy and Precision

Accuracy and precision calculated for the QC samples

during the intra- and inter –day run are given the (Table-

5). The intra-day (day-1) accuracy for Propranolol

ranged from 97.38- 100.38% while that of Diazepam

ranged from 97.18 – 100.04 %. The intra-day (day-1)

precision for Propranolol ranged from 0.14-0.57 % while

that of Diazepam ranged from 0.13-0.59%. The inter-day

(day-2) accuracy for Propranolol ranged from 96.74-98.70 % while that of Diazepam ranged from 97.56-

100.55%. The inter-day (day-2) precision for

Propranolol ranged from 1.39-3.60% while that of

Diazepam ranged from 0.98-1.95%. The results obtained

from intermediate precision (inter-day) also indicated a

good method precision. All the data were within the

acceptance criteria.

3.2.5 Specificity

Specificity was determined by comparison of the Blank

chromatogram with that of the Standard chromatogram

(Fig-4)

3.2.6 Room Temperature Stability

Stability studies were done for short term stability up to

12 hrs on the bench top for the MQC levels conditions.

Stability is calculated as the ratio of the mean peak area

of the stability sample to the mean peak area of the fresh

sample and expressed as the percentage (n=6). The room

temperature stability was found to be 101.63% for

Diazepam and 110.34 % for Propranolol. The results are

tabulated in Table-6.

3.2.7 Stress Degradation

Stress studies revealed that Diazepam is not susceptible

to degradation under acid, light (UV) and oxidative

stress conditions (Fig 5). However, in alkaline

conditions (0.1N NaOH), the drug was instable and the

degradation peak eluted earlier accompanied with a

drastic peak distortion and increased tailing. Except for



5

specificity of the analytical method to differentiate the

degradation peaks.

Stress studies on Propranolol indicated instability under

alkaline and photolytic conditions. This has been clearly

demonstrated by the help of overlap spectra of all the

stress samples as compared with that of freshly prepared

sample of similar concentration (Fig 5).

3.2.8 Robustness study

Robustness is the measure of method capacity to remain

unaffected by deliberate small changes in the

chromatographic conditions. The experimental conditions

were deliberately altered to evaluate the robustness of the

method. The impact of flow-rate (1.0 ± 0.1 ml/min), and

effect of mobile-phase composition (± 5%) on

chromatographic parameters such as retention time,

theoretical plates, and tailing factor, were studied. At

lower flow rate, the retention time of Diazepam was 8.10

± 0.04 minutes (n=6) while that of Propranolol was 2.4 ±

0.06 minutes. At lower flow rate, the tailing factor for

Diazepam and Propranolol remained unchanged. At higher flow rate, for both Diazepam and Propranolol

7.3and 2.4 ± 0.06 minutes tailing factor remained

unchanged as compared to normal flow. The elution was

earlier at higher flow rate. The retention time of

Diazepam and Propranolol were 8.3 ± 0.02 and 2.4 ±

0.03 minutes respectively (n=6) when the mobile phase

composed of 70 methanol and 30 parts of 20m

orthophosporic acid (pH 3.0).

3.3 Application of the method to dosage forms

The HPLC method developed is sensitive and specific for the quantitative determination of Diazepam and

Propranolol. The% assay of Diazepam in the tablet is

100.622 ± 0.06mg and % assay of Propranolol in the

tablet is 100.198± 0.11 mg. None of the tablets

ingredients interfered with the analyte peak. The

spectrum of Diazepam and Propranolol in the extracted

tablet was matching with that of standard compounds

indicating the purity of the compounds in the tablets.

alkaline conditions, the drug content was within 95 –105

% for all stress conditions indicating the stability and

Conclusions

The method gave accurate and precise results in the

concentration range of 2.50 – 50.00 µg/mL for

Diazepam and 1.00 to 20.00µg/mL for Propranolol. The

mobile phase composition consists of (65:35%v/v) of

Methanol and orthophosphoric acid (pH adjusted to 3.0

with glacial acetic acid), at the flow rate of 1.0 ml/min. The retention time of Diazepam is 7.72 ± 0.2 minutes

and that of Propranolol is 2.04 ± 0.2 minutes. The

column is Phenomenex 150 X 4.6mm, C18 column with

the particle size of 5µm. A rapid sensitive and specific

method for the simultaneous estimation of Diazepam

and Propranolol in the pharmaceutical tablet formulation

has been developed and validated.

Fig-3a: Linear calibration curve of Diazepam

Fig-3b: Linear calibration curve of Propranolol

y =

142,159.4546x +

47,042.4748

R² = 0.9962

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0.00 20.00 40.00 60.00

Series1

Linear

(Series1)

y =

161,887.2821x -

25,823.5299

R² = 0.9961

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0.00 10.00 20.00 30.00

Series1

Linear

(Series1)



6

Table 1. System Suitability test for Diazepam (above) and Proprtanolol (below)

DIAZEPAM

Sample ID Peak Retention Time Peak Area Theoretical Plates Tailing Factor

1 7.25 3735027 21320 1.07

2 7.27 3862485 21253 1.07

3 7.28 3905593 21241 1.07

4 7.28 3953105 21408 1.08

5 7.29 4052960 21414 1.07

6 7.28 3908860 21371 1.07

MEAN 7.275 3903005.0 21334.5 1.072

STDEV 0.0138 104781.65 75.70 0.0041

%CV 0.19 2.68 0.35 0.38

PROPRANOLOL

Sample ID Peak Retention Time Peak Area Theoretical Plates Tailing Factor

1 2.06 1796701 11182 1.5

2 2.07 1794265 11249 1.5

3 2.06 1798805 11185 1.51

4 2.06 1863036 11115 1.52

5 2.07 1914378 11183 1.51

6 2.06 1850305 11209 1.5

MEAN 2.063 1836248.3 11187.2 1.507

STDEV 0.0052 48472.69 43.72 0.01

%CV 0.25 2.64 0.39 0.54

Table 2. Sensitivity

DIAZEPAM

PROPRANOLOL

LOD

LOD

SR NO DRUG

SR NO DRUG

Retention Time Peak Area


1 7.36 18785

1 2.12 12757

2 7.36 18837

2 2.11 14369

3 7.37 20355

3 2.11 16250

MEAN 7.4 19325.7

MEAN 2.1 14458.7

ST DEV 0.01 891.81

ST DEV 0.01 1748.23

% CV 0.08 4.61

% CV 0.27 12.09

DIAZEPAM

PROPRANOLOL

LOQ

LOQ

SR NO DRUG

SR NO DRUG



1 7.37 15559

1 2.13 18292

2 7.34 17251

2 2.1 37075

3 7.36 13464

3 2.12 31926

MEAN 7.4 15424.7

MEAN 2.1 29097.7

ST DEV 0.02 1897.07

ST DEV 0.02 9705.66

% CV 0.21 12.30

% CV 0.72 33.36



7

Fig-4: Comparison of (a)Blank Chromatogram, (b) Propranolol alone (c) Diazepam alone and (d) sample

containing both Propranolol and Diazepam

a. Blank sample

b. Propronolol sample

c. Diazepam sample

d) sample containing both Propronolol and Diazepam

Table 3. Results of best-fit line for triplicate analysis for Propronolol and Diazepam

Diazepam

Curve Slope Intercept r2

1 14259.26 47,402.445 0.9962

2 14258.96 47,402.210 0.9964

3 14258.96 47,402.410 0.9963

Mean 14258.99 47,402.355 0.9963

Propronolol

Curve Slope Intercept r2

1 161887.29 25823.52 0.9961

2 161886.26 25823.52 0.9962

3 161887.29 25823.54 0.9963

Mean 161887.28 25823.52 0.9962

Table 4. Linearity and Range for Levosulpiride and rabeprazole demonstrating accuracy, carryover effect and



8

specificity of the method (Curve 1).

DIAZEPAM

SAMPLE ID Concentration

(Microgram/mL) Retention Time Peak Area Back Calc

Concentration % Accuracy

Blank 0 NA 0 NA

CC - 01 2.50 7.21 431518 2.70 108.18

CC - 02 10.00 7.23 1536982 10.48 104.81

CC - 03 20.00 7.24 2896571 20.04 100.22

CC - 04 30.00 7.27 4205821 29.25 97.51

CC - 05 35.00 7.27 4822057 33.59 95.97

CC - 06 40.00 7.30 5665085 39.52 98.80

CC - 07 45.00 7.3 6745582 47.12 104.71

CC - 08 50.00 7.31 7124797 49.79 99.57

Blank 0 NA 0 NA NA

• NA - Not applicable

NA - Not applicable

PROPRONOLOL

SAMPLE ID Concentration

(Microgram/mL)

Retention

Time Peak Area

Back Calc

Concentration % Accuracy

Blank 0.00 NA 0 NA 0

CC - 01 1.00 2.12 150256 1.09 108.77

CC - 02 4.00 2.12 705468 4.52 112.93

CC - 03 8.00 2.1 1197852 7.56 94.49

CC - 04 12.00 2.12 1800221 11.28 94.00

CC - 05 14.00 2.11 2283222 14.26 101.88

CC - 06 16.00 2.1 2550045 15.91 99.45

CC - 07 18.00 2.11 2879115 17.94 99.69

CC - 08 20.00 2.1 3282750 20.44 102.19

Blank 0 NA 0 NA NA



9

Table 5a. Results of inter and intra-day accuracy & precision for Diazepam

Nominal Concentration (µg/mL)

12.50 25.0 37.5

DAY 1

MEAN (n=6) 12.24 24.54 37.34

SD 0.12 0.46 0.53

% CV 0.98 1.87 1.42

DAY 2

MEAN (n=6) 12.24 24.54 37.71

SD 0.12 0.49 0.51

% CV 0.98 1.95 1.36

DAY 3

MEAN (n=6) 12.33 24.90 37.51

SD 0.15 0.49 0.51

% CV 1.21 1.95 1.36

Table 5b. Results of inter and intra-day accuracy & precision for Propronolol

Nominal Concentration (µg/mL)

5.00 10.00 15.00

DAY 1

MEAN (n=6) 5.02 9.94 14.66

SD 0.14 0.15 0.57

% CV 2.80 1.48 3.90

DAY 2

MEAN (n=6) 4.93 9.81 14.51

SD 0.15 0.14 0.52

% CV 2.94 1.39 3.60

DAY 3

MEAN (n=6) 4.85 9.79 14.41

SD 0.13 0.15 0.44

% CV 2.66 1.54 3.03



10

Fig-5a: Overlay Chromatogram showing the influence of various stress conditions on Propranolol;

Data 1- fresh sample Data2-Acid Stress, Data3 – Oxidative Stress; Data 4 –Photolytic Stress;

Data 5 – Alkaline Stress. Data 5 clearly indicates the spectral degradation of Propranolol due

to alkaline instability.

Fig-5b: Overlay Chromatogram showing the influence of various stress conditions on Diazepam; Data 1- fresh sample Data2-Acid Stress, Data3 – Oxidative Stress; Data 4 –Photolytic Stress; Data

5 – Alkaline Stress Data 5 clearly indicates the spectral degradation of Diazepam due to

alkaline instability.



11

Table 6a. Room Temperature Stability of Propronolol (n = 6).

PROPRONOLOL

FRESH SAMPLE

STABILITY SAMPLE

SR

NO

SAMPLE

ID

CONC

(µg/mL)

DRUG

SR

NO

SAMPLE ID CONC

(µg/mL)

DRUG

Rt PEAK

AREA

Rt PEAK

AREA

1 FRESH 25.00 2.15 988084

1 STABILITY 25.00 5.39 895502

2 FRESH 25.00 2.14 995208

2 STABILITY 25.00 5.38 960507

3 FRESH 25.00 2.14 972452

3 STABILITY 25.00 5.38 859319

4 FRESH 25.00 2.13 902062

4 STABILITY 25.00 5.37 843544

5 FRESH 25.00 2.13 957144

5 STABILITY 25.00 5.35 861571

6 FRESH 25.00 2.13 962213

6 STABILITY 25.00 5.34 945770

Mean

962860.50

Mean

894368.83

Stdev

33178.72

Stdev

48795.44

%

3.45

% Cv

5.46

% Stability 92.89

Table 6b. Room Temperature Stability of Diazepam (n = 6).

Diazepam

FRESH SAMPLE

STABILITY SAMPLE

SR NO SAMPL

E ID

CONC

(µg/mL

)

DRUG SR NO SAMPLE ID CONC

(µg/mL

)

DRUG

Rt PEAK

AREA

Rt PEAK

AREA

1 FRESH 25.00 2.12 3118390 1 STABILITY 25.00 5.35 2892382

2 FRESH 25.00 2.12 3154127 2 STABILITY 25.00 5.35 3121336

3 FRESH 25.00 2.12 3104326 3 STABILITY 25.00 5.34 2697060

4 FRESH 25.00 2.13 3080722 4 STABILITY 25.00 5.39 2847700

5 FRESH 25.00 2.14 3025992 5 STABILITY 25.00 5.41 2280303

6 FRESH 25.00 2.14 3206837 6 STABILITY 25.00 5.41 3006784

MEAN

3115065.67 MEAN

2807594.17

STDE

61947.98 STDE

295689.62

% CV

1.99 % CV

10.53

% Stability 90.13



12

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Documents

Research Article Vol: 1; Issue: 1 Development and Validation of ...ijpda.com/admin/uploads/kBjcGP.pdf · The injection volume is 20µL. The retention time of Diazepam and Propranolol