Available online www.ijpras.

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International Journal of Pharmaceutical Research & Allied Sciences, 2023, 12(2):108-115
https://doi.org/10.51847/qaTx5GtJZY

ISSN : 2277-3657
Original Article CODEN(USA) : IJPRPM

UV-Spectropectrophotometry for Determination of Diazepam by Comparative

Estimation of Methods of Calibration Curve and Reference Standard
Dobrina Tsvetkova1*, Stefka Ivanova2, Maria Chaneva3
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University-Pleven, Sofia 1000,
Bulgaria.
2
Bulgarian Scientific Society of Pharmacy, Bulgaria.
3
Clinic of Inrermnal Medicine, UMBALSM ”N. I. Pirogov” – Sofia, Bulgaria.

*Email: dtsvetkova@pharmfac.mu-sofia.bg
ABSTRACT

The objective of the current work was to compare the methods of the calibration curve (MCC) and the external
reference standard (MRS) for estimating the analytical parameters accuracy and precision in order to validate
the UV-spectrophotometric method for the determination of diazepam at λmax = 328 nm in 95% ethanol. The fact
that the detected absorbance at λmax = 328 nm, a particular wavelength for diazepam, could not be found in the
UV spectra of the blank solution was provided as evidence of selectivity. Analysis using linear regression was
performed on the experimental findings: y = 14387.x +0.05. An analysis of linearity was conducted by measuring
the linear regression coefficient: R2 > 0.998. LOD = 1.84.10–5 g/ml; LOQ = 1.04.10–5 g/ml. Accuracy was
reflected and determined by recovery degree R [%]  RSD [%] as per ICH guidelines: MCC: 100.49 %  1.35
%; MRS: 105.56 %  1.37 %. The fact that SD and RSD were less than 1.5 confirmed that the findings and actual
values were in agreement. From the precision assessment, all values for the content of Diazepam, obtained both
by MRS and MCC at the confidence possibility P = 98%, suit the relevant confidence interval: MCC: 4.88 mg 
4.96 mg; MRS: 5.12 mg  5.20 mg. Through the use of all of these data, it was demonstrated that the validated
approach was reliable and accurate for determining the dose of Diazepam in medication formulations.

Key words: Diazepam, Spectrophotometry, Validation, Accuracy, Precision

INTRODUCTION

Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one (Figure 1) is applied

commonly for therapy of features associated with alcohol, opiate, and benzodiazepine withdrawal, anxiety
and trouble sleeping [1]. The drug is used as miorelaxant [2] for muscle spasms and induced by stroke paresis [3].
Diazepam posesses effect on the growth of Chrysomya albiceps in rabbit carcass [4]. Diazepam is applied as an
anesthetic for preoperative premedication before endoscopic or surgical procedures like open-heart surgery [5] or
aortic valve implantation [6].
In European Pharmacopoeia [7] and British Pharmacopoeia [8] for the determination of Diazepam substance is
applied acid-base neutralization non-water method in the medium of acetic anhydride and titration with 0.1 M
perchloric acid by using indicator Nile blue [7] or potentiometrically [8] with Diazepam ion-selective electrode
[9]. For simultaneous potentiometric quantification of Diazepam, Bromazepam, and Clonazepam has been
described as the method with solid contact ion-selective electrodes [10]. Electrochemical assay of Diazepam in
pharmaceutical products by polarography has been reported [11].

© 2023 The International Journal of Pharmaceutical Research and Allied Sciences (IJPRAS). Open Access - This article is under the CC BY NC SA license
(https://creativecommons.org/licenses/by-nc-sa/4.0).
Tsvetkova et al. Int. J. Pharm. Res. Allied Sci., 2023, 12(2): 108-115

The advantages of the titrimetric method are the short time of analysis, quick and easy performance, cost-
effectivity, and simple instrumentation [12]. The disadvantage of titrimetric methods is the lack of selectivity for
the analysis of combinations in comparison with the separation techniques: gas chromatography, HPLC, and
capillary electrophoresis, which offer significant advantages, such as high separation efficiency, fast analysis, and
small sample volumes [13].
Diazepam dose estimation in tablet form using the RP-HPLC technique has been introduced [3]. For the
simultaneous measurement of Diazepam and Otilonium bromide in tablets, reversed-phase liquid chromatography
and capillary electrophoresis have been devised [14, 15].
Fluorimetry has been applied for the quantification of Diazepam in tablets and injections [16] and of other drugs
[17]. Spectrophotometric and fluorimetric methods for the assay of Diazepam, Bromazepam, and Clonazepam in
pharmaceutical and urine samples have been used [18].
The drawback of HPLC is that it requires a trained technician for monitoring. In comparison with HPLC, the
advantages of UV-spectrophotometric methods are simplicity of procedures and economy [19].
The following spectrophotometric techniques for analyzing Diazepam have been published:
1. first-order derivative UV-spectrophotometry for determination of Diazepam alone [20] and for simultaneous
quantification in combination with Diazepam and Otilonium bromide [21],
2. second-order-derivative spectrophotometry for analysis of mixtures of 1,4-benzodiapines [22],
3. ratio-spectra derivative spectrophotometry for simultaneous assay of Diazepam and Otilonium bromide [23],
4. visible spectrophotometry for Diazepam in pure form, tablets, and ampoules after the interaction with picric
acid at λ = 475 nm and with 3,5-dinitrobenzoic acid and 2,4-dinitrobenzoic acid at λ = 500 nm [24].

Mass spectrometry is also used for drug analysis [25]. Derivative spectrophotometry has the drawback of being
sensitive to adjustments in the settings of the device. Particularly in the zero-crossing approach, which lacks
repeatability due to inaccuracies in the registration of the spectrum, small variations in the wavelength setting
have a significant impact on the outcome [26]. Chemometric assisted spectrophotometric method is applied for
the simultaneous evaluation of drugs, such as of Amlodipine Besylate and Candesartan Cilexeti [27]. Contrary to
the UV-derivative approach, the standard UV method has lower sensitivity to changes in the apparatus settings
and is one of the most widely used methods for pharmaceutical analysis because it is quick, easy, specific,
accurate, and speedy [19].
Due to the content chemical structure of chromophore groups, Diazepam is developed UV-spectrophotometric
methods for direct determination in the ultraviolet region without the need for a derivatization reaction. UV-
spectrophotometric method for analysis of Diazepam substance in solvent mixture methanol: distilled water = 1:
1 at λ = 231 nm has been described [26]. UV-spectrophotometric methods for quantification of Diazepam and
Sodium benzoate in dosage drug forms at λ = 306 nm [28] and for simultaneous assay of Diazepam, Caffeine, and
Phenylpropanolamine hydrochloride in tablets have been developed [29].
Following British Pharmacopoeia the determination of Diazepam in tablets is spectrophotometrically in UV-area
in 0.5 % methanolic sulphuric acid a by method of specific absorbance: A(1%, 1 cm) = 450 at λ = 284 nm [7].
By estimating analytical characteristics such as selectivity, linearity, LOD, LOQ, accuracy, and precision, the
current study aimed to validate and compare traditional UV-spectrophotometric techniques of calibration curves
and methods of external standards for the measurement of Diazepam in ethanol.

MATERIALS AND METHODS

Materials
1. Reference standard (RS): Diazepam.
2. Purity-graded chemicals for analytical use: SZBD 0500 V UN 1170, Sigma Aldrich, 95% ethanol.

Method. UV-Spectrophotometry

1. Equipment.
UV-VIS diode array spectrophotometer (Hullett Packard N: 8452 A).

2. Preparation of blank solution for estimation of an analytical parameter selectivity.

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To estimate the analytical parameter selectivity, a blank solution in 95% ethanol was made. Diazepam-free
supplement starch, which is utilized in tablet manufacturing, was added to blank solutions. A volumetric flask
was used to dissolve 0.05 g of supplement starch that had been precisely weighed in 95% ethanol to a volume of
25.0 ml, and a portion of the solution equal to 1.0 ml was divided into 10.0 ml and then diluted again in 95%
ethanol. 95% ethanol was applied as a blank solution. The data for the absorbance were measured at λ = 328 nm.

3. Preparation of solutions of reference standard Diazepam in 95% ethanol for estimation of analytical
parameter linearity.
95% ethanol was used to dissolve a precisely measured amount of the reference standard Diazepam (25 mg, 37.5
mg, 50 mg, 75 mg, 100 mg, 125 mg, and 175 mg), and the same solvent was then used to dilute the solution to
250.0 ml in volumetric flasks. Aliquot portions of 10.0 ml of the obtained solutions were diluted in 95% ethanol
to 100.0 ml in volumetric flasks, resulting in solutions with equivalent concentrations of diazepam: 1.10 –5 g/ml,
1.5.10–5 g/ml, 2.10–5 g/ml, 3.10–5 g/ml, 4.10–5 g/ml, 5.10–5 g/ml and 7.10–5 g/ml. All solutions were analyzed at λ
= 328 nm against 95% ethanol and the absorbances were measured.

4. Preparation of model mixture with Diazepam for validation of the UV-method for analytical parameters
accuracy and precision (repeatability).
By adding an active ingredient, Diazepam, equal to 100% (5 mg) of the theoretical concentration of Diazepam in
tablets (5 mg), a model combination was created from the supplement in tablet form (starch). The model
combination weighed 0.05 g on average. The model mixture amount, which is equal to 5 mg of benzodiazepine,
was precisely measured to create six identical, homogeneous samples. All samples were dissolved in 95% ethanol
and were diluted with 95% ethanol to 25.0 ml in volumetric flasks. The produced solutions were diluted in
volumetric flasks with aliquots of 1.0 ml each in 95% ethanol to 10.0 ml. As the blank solution, 95% ethanol was
used to measure the absorbances of the produced solutions at λ = 328 nm.

5. Preparation of reference solution of Diazepam for the method of an external standard.

In volumetric flasks, 95% ethanol was used to dilute a precisely measured quantity of 5 mg of the reference
standard benzodiazepine to 25.0 ml. Diazepam solutions with various concentrations were created by diluting
aliquot portions of the resultant solution, which included 1.0 ml, in 95% ethanol to 10.0 ml in a volumetric flask:
2.10–5 g/ml. The absorbance of the final solution was detected at λ = 328 nm applying 95% ethanol as
compensation.

6. Root limit mean square error method (RMSE) for the determination of limit of detection (LOD) and limit of
quantitation (LOQ).
The examination of solutions with low concentrations was used to create calibration curves. After the data
underwent linear regression analysis, the linear correlation coefficients (R2) were discovered. The regression
equation: y = a.x + b was applied for obtaining the data for the predictable absorbance (Ap); the error E = |Ap -

A|; E2 = [|Ap - A|]2, E1=

E  ; RMSE = E1 , LOD = 3.RMSE/а, LOQ = 10.RMSE/а [30].
n−2

RESULTS AND DISCUSSION

Validation of UV-spectrophotometric method [31-34]

1. Estimation of analytical parameter selectivity
When a component that could be present in the sample matrix is present, the analyte is quantitatively
detected by selectivity [35, 36]. Selectivity was demonstrated by the absence of measurable absorption at the
targeted wavelengths in the UV spectra of blank solutions.

2. Investigation of analytical parameter linearity, accuracy and precision.

Application of the method of linear regression analysis
Linearity is the portion of a detector-derived signal that continues to exhibit a linear relationship with the analyte's
concentration [31-35].
A series of solutions with increasing concentrations (1.10-5 g/ml ÷ 7.10-5 g/ml) were created in order to investigate
the linearity of analytical parameters from the reference standard Diazepam. For the investigation of analytical

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parameter accuracy and repeatability 6 mixtures containing Diazepam were prepared. The absorbances (A) at
wavelength λ = 328 nm were measured and UV-spectra were detected and were demonstated on Figure 1.

Linearity Accuracy and precision

a) b)
Figure 1. UV-spectra for standard solutions of Diazepam.

Table 1 provides an overview of the experimental findings for the observed absorbances of a number of solutions
with escalating Diazepam doses.

Table 1. Concentrations and absorbances for reference standards of Diazepam in 95% ethanol for estimation of
analytical parameter linearity.
N: C [g/ml] A
1. 1.10–5 0.18515
2. 1.5.10–5 0,27440
3. 2.10–5 0.32350
4. 3.10–5 0.49339
5. 4.10–5 0.61980
6. 5.10–5 0.78979
7. 7.10–5 1.0435

The obtained experimental results for absorbances of reference standards of Diazepam were used to linear
regression analysis. The regression equation's parameters, which showed a linear connection between absorbances
and concentrations at the relevant concentration intervals, were given in Table 2.

Table 2. Parameters of the regression equation for Diazepam.

N: Parameter Result
1. The linear interval [g/ml] 1.10–5 ÷ 7.10–5
2. Regression equation y = 14387.49. x + 0.05
3. Slope (a) 14387.49
4. Standard slope error 286.9014
5. Inrersept (b) 0.049782
6. Standard intercept error 0.011178
7. Correlation coefficient (R2) 0.9980

Figure 2 depicts the calibration curve, which demonstrates the linear connection between A and concentration C
[g/ml]. Linear regression coefficients serve as a measure of linearity: R2  0.9980.

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Diazepam Linearity
1.2
y = 14387,49 . x + 0,05
1.0435
1 R² = 0,9980

Absorbance [A U]
0.8 0.78979

0.6 0.6198
0.49339
0.4
0.3235
0.2744
0.2 0.18515

0
0 0.00002 0.00004 0.00006 0.00008

Concentration [g/ml]
Figure 2. Calibration curve for linearity for Diazepam.

3. Estimation of analytical parameters limit of detection (LOD) and limit of quantitation (LOQ).
Table 3 includes the findings from the examination of standard solutions containing progressively higher
concentrations of Diazepam to investigate LOD and LOQ: C [g/ml] – concentration, A – measured absorbance,
Ap – calculated by calibration curve absorbance, Е = |Ap – A|, Е2 = [|Ap – A|]2, RMSE = E1 , LOD = 3.RMSE/а,
LOQ = 10.RMSE/а. LOD and LOQ are based on the regression equation: y = 14387.49. x + 0.05 by application
of RMSE-method [30].

Table 3. RMSE method for LOD and LOQ for Diazepam in 95% ethanol.
N: C [g/ml] A Ap E = |Ap – A| Е2 = [|Ap –A|]2
1. 1.10–5 0.18515 0.19387 0,00872 0.00008
2. 1.5.10–5 0,27440 0.26581 0,00859 0.00007
3. 2.10–5 0.32350 0.33775 0,01425 0.00020
4. 3.10–5 0.49339 0.48162 0,01177 0.00014
5. 4.10–5 0.61980 0.62550 0,00570 0.00003
6. 5.10–5 0.78979 0.76937 0,02042 0.00042
7. 7.10–5 1.04350 1.05712 0,01362 0.00019
E1=  E  = 0.000226
 E 2 = 0.00113 n−2
RMSE= 0.000226 = 0.015

LOD = (3.0.015)/14387.49 = 3.13.10-6 g/ml LOQ = (10.0.015)/14387.49 = 1.04.10-5 g/ml

4. Estimation of analytical parameters accuracy and precision for Diazepam.

Table 4 are presented values for: 1) included content of Diazepam; 2) weighed quantity of Diazepam for analysis;
3) absorbances: A, ASt = 0.32350.

Table 4. Added and content aud absorbances for mixtures of Diazepam.

N: Added content [mg] Weight content [g] Absorbance A
1. 4.84 0.0484 0.32530
2. 4.86 0.0486 0.32675
3. 4.88 0.0488 0.32687
4 4.91 0.0491 0.32707
5. 4.92 0.0492 0.32709
6. 4.94 0.0494 0.32747
̅
X 0.32676
SD 0.0008
RSD [%] 0.24

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By using the calibration curve technique (MCC) and the external reference standard method (MRS) in Table 5,
the amount of Diazepam was determined. It is indicated as N – number of the individual measurements (n = 6);
C – received content of Diazepam; UC – Schöveneous value for obtained quantity (UС); R (%) – level of
recovery (RC); X̅ – arithmetical mean; SD – standard deviation; RSD [%] – relative standard deviation; SX
̅– mean
̅– t.SX
quadratic error; X ̅X
̅+ t.SX̅– confidence interval (CI); E (%) – relative error. For the calculation of the
confidence interval are used: the confidence probability was 98% and the coefficient of Student was 3.37.

Table 5. Obtained quantity (C), recovery (R), and Schöveneous criterion (U) for C –estimation by methods of
the calibration curve and of external standards.
Method of the calibration curve Method of external standard
Obtained quantity RC Obtained quantity
N: UС R [%] UС
С [mg] [%] С [mg]
1. 4.94 102.07 0.67 5.19 107.23 1.00
2. 4.95 101.85 1.00 5.20 107.00 1.33
3. 4.93 101.02 0.33 5.18 106.15 0.67
4. 4.90 99.80 0.67 5.15 104.89 0.33
5. 4.89 99.39 1.00 5.14 104.47 0.67
6. 4.88 98.79 1.33 5.12 103.64 1.33
̅ SD
X 4.92  0.03 5.16  0.03
R [%]  RSD [%] 100.49 1.35 105.56 1.37
SD 0.03 1.36 0.03 1.45
RSD 0.61 1.35 0.58 1.37
̅
SX 0.012 0.56 0.012 0.59
̅
t.SX 0.04 1.89 0.04 1.99
̅X
̅– t.SX
X ̅+ t.SX
̅ 4.88  4.96 98.60 102.38 5.12  5.20 103.57 107.55
Е [%] 0.24 0.56 0.23 0.56

The data for Chauvenet's criteria are lower for all of the observed experimental findings than the highest permitted
standard requirement: U < 1.73 (n = 6) is used to determine if findings with sharp discrepancies should be
eliminated.

Accuracy
A sample standard deviation (SD) is determined for the estimation of accuracy and precision by using Bessel's
adjustment.
Accuracy is the level of agreement between the mean result of the repeated measurements and the real values. By
carrying out the recovery for 6 samples at a level equal to 100% of the nominal concentration, the accuracy of the
procedure was assessed. R [%]  RSD [%] was used as a measure of accuracy in accordance with ICH
recommendations [31-34]: MCC: 100.49 %  1.35 %; MRS: 105.56 %  1.37 %. Results indicate that all data for
degree of recovery fit respective confidence intervals at the specified confidence level P = 98%: MCC: 98.60 %
 102.38 %; MRS: 103.57 %  107.55 %. SD and RSD were less than 1.5, demonstrating agreement between the
findings and real values.

Precision (repeatability)
The uncertainty of the outcome, which is calculated by the SD, RSD, and CI [30-36], is employed for the estimate
of an analytical parameter with accuracy (repeatability). By scanning samples of Diazepam (n = 6) repeatedly,
repeatability was achieved. It is clear from the assessment of precision that all values for the content ofDiazepam,
obtained by MES and MCC at the confidence level P = 98%, are consistent with the applicable confidence interval:
MCC: 4.88 mg  4.96 mg; MRS: 5.12 mg  5.20 mg. Due to the close proximity of the findings to the average
value and the corresponding small confidence interval, all values for SD are less than SD = 1.5, indicating good
accuracy.

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CONCLUSION

The current UV-spectrophotometric technique was validated in accordance with the ICH criteria for the analytical
parameters of selectivity, linearity, LOD, LOQ, accuracy, and precision for the measurement of diazepam in 95%
ethanol using the method of the calibration curve and the methodology of the external standard. The consistency
of the results for repeatability and accuracy within the appropriate confidence intervals demonstrated that the
validated technique was determined to be precise and accurate for use in determining the amount of diazepam in
the dose medication formulation.

ACKNOWLEDGMENTS : None

CONFLICT OF INTEREST : None

FINANCIAL SUPPORT : None

ETHICS STATEMENT : None

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