Original Article

Quantitative determination of dexamethasone sodium

phosphate in bulk and pharmaceuticals at suitable pH
values using the spectrophotometric method
Mohammed Fanokh Al‑Owaidi,
Sura L. Alkhafaji, Abstract
Abdulbari Mahdi Mahood
Dexamethasone sodium phosphate (DSP) is an ester of dexamethasone with
Department of Pharmaceutical anti‑inflammatory action. This study provides new insights to develop a simple,
Chemistry, College of Pharmacy,
University of Kerbala, Kerbala, Iraq precise, and accurate spectrophotometric method for the quantitative determination
of DSP in bulk and pharmaceuticals. The method was validated before being applied
J. Adv. Pharm. Technol. Res.
to determine the DSP in six pharmaceutical injection forms from different companies.
DSP is soluble in phosphate buffer, so it was used as a solvent, and a pH of 6 was
found to be suitable for determination purposes. The DSP solution was scanned in the
ultraviolet range (200–400 nm) using a double‑beam spectrophotometer with a 1‑cm
quartz cell. The wavelength (λ max) of DSP was set at 242.5 nm, following the Beer–
Lambert law for concentrations from 2 to 50 µg/ml. Dexa AIWA (Germany) showed the
best results, being very close to the bulk value with no significant variation. Similarly,
Dexamed (Cyprus) and HEMAZON (Syria) showed no significant differences from the
bulk; however, the three remaining injections, DEXAKAL (India), DEXABRU (India),
and DEXARON (China), showed significant variations from the bulk. Estimated limit of
detection and limit of quantitation values for DSP were 0.83 and 2.5 µg/ml, respectively,
with a regression coefficient of 0.999. Recovery studies were then used to determine
the accuracy of the suggested method. The percentage of recovery was found to be
98.58%–102.52%. All results are suggesting a pivotal method for the routine analysis
of DSP both in pure form and the commercially pharmaceutical forms.

Key words: Dexamethasone sodium phosphate, phosphate buffer, ultraviolet

spectrophotometry, validation

INTRODUCTION

Dexamethasone sodium phosphate (DSP) is an inorganic

Address for correspondence: ester of dexamethasone that is used to treat inflammatory,
Dr. Sura L. Alkhafaji, allergy, endocrine, rheumatic, dermatologic, and others. It
Department of Pharmaceutical Chemistry, College of Pharmacy, is also used in a majority of chemotherapy patients.[1]
University of Kerbala, Hai Al‑Muadhafeen Campus, 56001
Kerbala, Iraq.
E‑mail: [email protected] Chemically, DSP is a pregna‑1,4‑diene‑3,20‑dione,
9‑fluoro‑11,17‑dihydroxy‑16‑methyl‑21‑(phosphonoooxy)‑,
Submitted: 06‑Mar‑2021 Revised: 30-Jun-2021
Accepted: 19-Jul-2021 Published: 20-Oct-2021
This is an open access journal, and articles are distributed under the terms
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Access this article online License, which allows others to remix, tweak, and build upon the work
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Website:
www.japtr.org For reprints contact: [email protected]
How to cite this article: Al-Owaidi MF, Alkhafaji SL, Mahood AM.
Quantitative determination of dexamethasone sodium phosphate
DOI:
in bulk and pharmaceuticals at suitable pH values using
10.4103/japtr.japtr_6_21 the spectrophotometric method. J Adv Pharm Technol Res
2021;12:378-83.

378 © 2021 Journal of Advanced Pharmaceutical Technology & Research | Published by Wolters Kluwer - Medknow
 Al‑Owaidi, et al.: Spectrophotometric determination of dexamethasone sodium phosphate in bulk and pharmaceuticals

disodium salt, (11 β, 16 α) with the chemical formula of Materials

C22H28FO8PNa2 [Figure 1]. DSP generally appears as a DSP (100% purity) was obtained from Samarra Drug
white‑to‑creamy white powder with a molecular weight Industry; this was used as a reference standard. Monosodium
of 516.41 g/mol. It is excessively hygroscopic, with a water phosphate (NaH2PO4.2H2O) (99% purity) was provided
solubility of 1.52 mg/ml, and its solutions have pH values by the Gainland Chemical Company, UK, while HiMedia
between 7 and 8.5 with pKa of 1.89.[2] Laboratories, India, provided disodium phosphate (Na2HPO4)
and sodium hydroxide (NaOH) (each of 99% purity). The
DSP penetrates the central nervous system and is commercial dosage forms of DSP from six different companies
metabolized in the liver, being mainly eliminated in the were all injections of 8 mg/2 mL. These dosages were bought
urine.[3] Unbound dexamethasone crosses cell membranes from the local market after checking both manufacturing
and binds with a great affinity to specific cytoplasmic and expire dates, and took the form Dexa AIWA® (T and D
glucocorticoid receptors. The forming complex crosses Pharma GmbH, Germany), DEXABRU (Brawn Laboratories
the nuclear membrane and modulates the gene‑mediated Limited, India), DEXAKAL (Khandelwal Laboratories
protein production. Dexamethasone’s anti‑inflammatory Pvt. Ltd., India), Dexamed (Medochemie Ltd., Limassol,
effects are assumed to be due to phospholipase A2 inhibitory Cyprus), DEXARON® (Shanghai Pharm. Co., Ltd., China),
proteins called lipocortins, which regulate the manufacture and HEMAZON (Ibn Hayyan Pharm., Syria).
of potent inflammatory mediators such prostaglandins and
leukotrienes.[4]
Preparation of stock solutions
Dexamethasone sodium phosphate solution (100 µg/mL)
The working solution was prepared by taking 0.01 g of DSP
Literature shows various methods have been developed to
and dissolving it in 10 ml of distilled water (DW); then, the
estimate DSP. These methods include spectrophotometry,[5,6]
solution was diluted with DW to 100 mL.
kinetic spectrophotometry,[7] liquid chromatography,[8‑10]
high‑performance liquid chromatography (HPLC),[11,12]
Sodium dihydrogen phosphate and disodium hydrogen phosphate
HPLC with mass spectrometry (HPLC/MS), [13,14]
solutions
reversed‑phase HPLC in combination with other drugs,[15,16]
A 7.8 g of sodium dihydrogen phosphate (NaH2PO4) and
and electrochemical methods.[17]
7.10 g of Na2HPO4 were accurately weighed and transferred
into a 250‑ml separate graduated volumetric flask and
This study used a mixed‑methods approach based on
solubilized in 50 and 100 ml of DW, respectively. The
ICH guidelines for assessing DSP in the injection dosages solutions were then made up with DW to achieve a solution
and then to be checked afterward. The approach proposes of 0.2 M each of NaH2PO4 and Na2HPO4.
a new methodology for assessing DSP uses well‑known
generic products to develop a cheap, sensitive, and Preparation of buffer solutions
effective method for the quantitative determination of Buffers with various pH values (2, 3, 4, 6, 6.4, 7, and 8) were
DSP in pure and pharmaceutical in a suitable buffer as prepared using NaH2PO4 solution and Na2HPO4 solution
a solvent. in different proportions, as shown in Table 1; then, 0.1 M
HCl and 0.1 M NaOH solution were used to adjust the pH
MATERIALS AND METHODS of solutions and measured using the pH meter device.

Instrumentation Determination of dexamethasone spectrum

An ultraviolet (UV)‑visible double‑beam spectrophotometer After dilution of standard drug solutions with different
UV‑1800, with two 1‑cm quartz cells (Shimadzu UV buffers, the solutions containing 40 μg/ml of DSP were
spectrophotometer, Japan), a pH meter (Hanna, Romania),
pipettes of various volumes, and a digital electronic Table 1: Solution amounts used to prepare
balance (Denver, Germany) were used in this study. various pH values
pH value NaH2PO4 solutions Na2HPO4 solutions
(0.2 M) (mL) (0.2 M) (mL)
2 0.66 11.8
3 21.9 3.1
4 1.32 23.67
6 43.85 6.15
6.4 36.75 13.25
7 19.5 30.5
8 2.65 47.35
NaH2PO4: Sodium dihydrogen phosphate, Na2HPO4: Disodium hydrogen
Figure 1: Chemical structure of dexamethasone sodium phosphate phosphate

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 Al‑Owaidi, et al.: Spectrophotometric determination of dexamethasone sodium phosphate in bulk and pharmaceuticals

scanned from 200 to 400 nm to select the maximum Procedure for sample preparation
wavelength (λ max). The solution shows maximum Each injection solution (8mg/ mL) was transferred to 100 mL
absorption at 242.5 nm. volumetric flask and diluted using a previously prepared
buffer of pH 6; in each case, the flask was standardized by
Selection of suitable pH adding the buffer first. After that, for each product, three
A 10 µg/ml standard solution of DSP was prepared different concentrations of the drug (5, 10, and 15 µg/ml)
were prepared to estimate absorbance accurately, with a
using 1 mL of 100 µg/ml stock solution; this was then
control sample consisting of just the buffer solution at pH 6.
transferred into a series of 10‑ml graduated volumetric
flasks. Then, the volume was made up to 10 ml with one RESULTS AND DISCUSSION
of the buffers; for each buffer value, two samples were
prepared along with a control flask. The samples were Selection of suitable pH
scanned using the spectrophotometer to measure the The highest and lowest levels of pH were determined based
absorbance of DSP at the λ max (242.5 nm). The highest on their pKa values (1.8 and 6.4). Consequently, at lower
absorbance appeared at pH 6, and this pH was selected pH (<3), DSP becomes uncharged, while when the pH
for the preparation of the calibration curve. The data are increases, it takes on anionic forms as monoanionic and
summarized in Table 2. dianionic dexamethasone phosphate (DSP−, DSP − 2). This
means the reaction is dependent on pH value. In terms of
spectroscopy, this is reflected in the transfer of electrons
Table 2: Absorbance of 10 µg/ml dexamethasone
between different energy levels, such as the move from the
sodium phosphate at various pH levels
nonbonding orbital sigma (σ) to the sigma (σ*) antibonding
pH value Absorbance Mean orbital, which reverses higher pH values. This could explain
Sample number 1 Sample number 2 the higher absorbance value at pH = 6 [Table 2].[18,19]
2 0.248 0.266 0.257
3 0.259 0.261 0.260 Selection of wavelength
4 0.268 0.265 0.267 With the reference solution, a UV spectroscopic scanning
6 0.261 0.276 0.268 run between 200 and 400 nm that was performed to
6.4 0.250 0.256 0.253 determine the optimal UV wavelength (maximum) for
7 0.255 0.266 0.260 detection of DSP. Therefore, 242.5 nm was selected as the
8 0.250 0.259 0.254 working wavelength for DSP, as shown in Figure 2.

Table 3: Calibration data for dexamethasone Validation

sodium phosphate Linearity and calibration curve
The stock solution was diluted with a buffer (pH = 6) to
Concentration (µg/ml) Absorbance
2 0.051
4 0.101
6 0.150
8 0.224
10 0.266
15 0.398
20 0.526
25 0.654
30 0.784
35 0.875
40 0.999
45 1.135
Figure 2: Spectrum of dexamethasone sodium phosphate
50 1.260 (40 µg/ml) at pH = 6

Table 4: Accuracy and precision in standard solution

Concentrations Concentrations RE (%)* Recovery (%)* SD* RSD (%)*
taken (µg/ml)* found (µg/ml)*
15 15.084 0.56 100.65 0.380 2.52
30 30.802 2.67 102.67 0.141 0.46
45 44.902 0.21 99.7 0.040 0.091
*Mean of four replicates. RE: Relative error, SD: Standard deviation, RSD: Relative SD

380 Journal of Advanced Pharmaceutical Technology & Research | Volume 12 | Issue 4 | October‑December 2021
 Al‑Owaidi, et al.: Spectrophotometric determination of dexamethasone sodium phosphate in bulk and pharmaceuticals

make a series of DSP standard solution concentrations recovery were found to be 1.15% and 101.0%, respectively, while
ranging from 2 to 50 µg/ml. Absorbances were determined. the mean RSD% was 1.024%. The results summarized in Table 4
By plotting the absorbance versus concentrations, the confirmed that the method used was accurate and precise.
calibration curve was constructed and regression equation
was intended. Regarding the curve shown in Figure 3, the Detection limit and quantification limit
linear equation was y = 0.025x + 0.0132 and the correlation Following ICH guidelines, limit of quantitation (LOQ) and
coefficient (r2) was 0.9999 which is indicated a good linearity. limit of detection (LOD) were estimated as 3 SD/slope and 10
The calibration data are shown in Table 3. SD/slope, respectively, where SD is the standard deviation
of the intercept. The LOD was 0.6371 µg/ml and the LOQ
Accuracy and precision was 1.930 µg/ml in DSP for five replicate determinations.
The accuracy of the method was represented by percent relative A summary of the validation parameters is shown in Table 5.
error (RE%) while the precision was represented by the relative
standard deviation (RSD%). The accuracy (recovery) and the Determination of active dexamethasone sodium phosphate in injection
precision were thus estimated for a series of four replicates dosage forms in the Iraqi market
of three concentration levels of the standard solution (15, 30, The summarized analysis results, shown in Table 6,
and 45 µg/ml). The percentage of recovery, RE%, and RSD% indicated a high percentage of recovery with low RSD%, and
were estimated for each sample. The mean RE% and the mean indicated that the method is applicable for routine analysis
of pharmaceutical forms.
Table 5: Validation parameters for
dexamethasone sodium phosphate using the
1.4
proposed method y = 0.025x + 0.0132
1.2 R² = 0.999
Parameters DSP
Linearity (µg/mL) 2-50 1
Absorbance

Wavelength (nm) 245.5 0.8

Slope (m) 0.025
0.6
Intercept 0.0132
Correlation coefficient (r2) 0.999 0.4
LOD (µg/mL) 0.6371 0.2
LOQ (µg/mL) 1.930
0
Linear equation y=0.025x+0.0132 0 10 20 30 40 50 60
RSD (%) 1.024 Concentrations (µg/ml)
DSP: Dexamethasone sodium phosphate, RSD: Relative standard deviation, LOD:
Limits of detection, LOQ: Limit of quantification Figure 3: Calibration curve for dexamethasone sodium phosphate

Table 6: Accuracy and precision of dexamethasone sodium phosphate determination in injections

Trade name/company Taken (µg/mL)* Found (µg/mL)* RE (%)* Recovery (%)* SD* RSD (%)*
DEXARON (China) 5 4.818 0.017 95.8 0.082 1.72
10 9.512 0 95.12 0.422 4.447
15 14.298 0.005 95.32 0.240 1.685
Dexa AIWA (Germany) 5 4.912 −0.0033 98.24 0.104 2.137
10 9.685 0.0033 96.85 0.146 1.517
15 14.992 0.00667 99.94 0.078 0.526
DEXA KAL (India) 5 4.7 0.018 95.30 0.099 2.112
10 9.352 0.004 93.52 0.069 0.740
15 13.77 0.014 91.85 0.100 0.7305
HEMAZON (Syria) 5 5.058 0.044 101.16 0.121 2.41
10 9.352 0 93.52 0.119 1.283
15 14.445 0.006 96.3 0.160 1.114
DEXABRU (India) 5 4.698 0.046 93.96 0.099 2.124
10 9.298 0.021 92.98 0.023 0.248
15 13.618 0.015 90.78 0.151 1.111
Dexamed (Cyprus) 5 5.365 0.14 107.26 0.100 1.877
10 9.978 0.02 99.78 0.122 1.224
15 14.832 −0.0123 98.88 0.211 1.426
*Mean of four replicates. SD: Standard deviation, RSD: Relative SD

Journal of Advanced Pharmaceutical Technology & Research | Volume 12 | Issue 4 | October‑December 2021 381
 Al‑Owaidi, et al.: Spectrophotometric determination of dexamethasone sodium phosphate in bulk and pharmaceuticals

Table 7: t‑test data 110

Companies Unpaired t‑test 105

% Drug Content
P P 100
summery
95
Bulk versus DEXAKAL (India) 0.0056 **
Bulk versus DEXARON® (China) 0.0008 *** 90
Bulk versus Dexa AIWA® (Germany) 0.3611 NS 85
Bulk versus Dexamed (Cyprus) 0.3828 NS 80

DEXAKAL
(India)

DEXARON®

AIWA®
(Germany)

Dexamed

HEMAZON
(Syria)

DEXABRU
(India)
(China)
Bulk

Dexa
(standard)

(Cyprus)
Bulk versus HEMAZON (Syria) 0.3579 NS
Bulk versus DEXABRU (India) 0.0026 **
DEXAKAL (India) versus 0.1417 NS
DEXARON® (China) Companies
DEXAKAL (India) versus Dexa 0.0232 *
AIWA® (Germany) Figure 4: Relative content of dexamethasone sodium phosphate in
DEXAKAL (India) versus 0.0413 * comparison to the bulk
Dexamed (Cyprus)
DEXAKAL (India) versus 0.2325 NS three‑star (***) level, which represents P < 0.001, resulting
HEMAZON (Syria) in the injection’s drug content differing from that of the
DEXAKAL (India) versus 0.5125 NS bulk chemical.
DEXABRU (India)
DEXARON® (China) versus Dexa 0.0329 * Comparing the company products with each of the
AIWA® (Germany)
others shows that DEXA KAL and DEXARON are closely
DEXARON® (China) versus 0.0695 NS
Dexamed (Cyprus) matched, with no significant differences between them.
DEXARON® (China) versus 0.5198 NS In the similar manner, there are no significant differences
HEMAZON (Syria) and approximately equivalent drug content among
DEXARON® (China) versus 0.0418 * the following products: DEXAKAL and HEMAZON;
DEXABRU (India) DEXAKAL and DEXABRU; DEXARON and Dexamed;
Dexa AIWA® (Germany) versus 0.2649 NS DEXARON and; Dexa AIWA and Dexamed; Dexa AIWA
Dexamed (Cyprus) and; Dexamed and HEMAZON; and HEMAZON and
Dexa AIWA® (Germany) versus 0.6045 NS DEXABRU. However, significant differences could observe
HEMAZON (Syria)
between other companies products' such as between DEXA
Dexa AIWA® (Germany) versus 0.0113 *
DEXABRU (India) KAL and Dexa AIWA, where a comparison generates a one
Dexamed (Cyprus) versus 0.2245 NS star (*) significant, representing P < 0.05, which indicates the
HEMAZON (Syria) differences in the drug content of the injections produced
Dexamed (Cyprus) versus 0.0289 * by these companies. Similar differences can also be seen
DEXABRU (India) between DEXA KAL and Dexamed; DEXARON and DEXA;
HEMAZON (Syria) versus 0.1421 NS DEXARON and DEXABRU; Dexa AIWA and DEXABRU;
DEXABRU (India) and Dexamed and DEXABRU (India). All result data are
*Signifies P<0.05, **Signify P<0.01, ***Signify P<0.001. NS signifies P>0.05.
NS: No significance reported in Table 7 and Figure 4.

Statistical analysis of the results of dexamethasone CONCLUSION

sodium phosphate commercial dosages
Statistical analysis using a t‑test showed a set of significant The findings of this study show that the UV approach can
differences between the products. Dexa AIWA offered the be utilized for routine analysis of DSP in bulk formulations,
best results, being very close to the bulk with no significant as well as for the analysis of marketing injections. It is ideal
variations. This was followed by Dexamed and HEMAZON, for the intended application, especially in forensic science
which also showed no significant differences in comparison laboratories and other pharmaceutical analysis laboratories.
with the bulk. By comparing a series of the pharmaceutical preparations from
different companies with the bulk, Dexa AIWA® (Germany),
However, DEXA KAL and DEXABRU showed significant Dexamed (Cyprus), and HEMAZON (Syria) were found
differences from the bulk. Thus, these forms showed to be close to the bulk, offering reliable drug content
a significant variation at the two‑star level (**), which levels. However, DEXAKAL (India), DEXABRU (India),
represents P < 0.01, suggesting that the drug content of and DEXARON® (China) were found to have significant
these Indian brands was lower than that of the bulk, while differences in terms of variation in drug content as compared
DEXARON showed a very significant variation at the with the bulk.

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 Al‑Owaidi, et al.: Spectrophotometric determination of dexamethasone sodium phosphate in bulk and pharmaceuticals

Financial support and sponsorship determination of dexamethasone and fluoroquinolones; in vitro

Nil. study. S Afr J Chem 2019;72:130‑5.
11. Prakash K, Sireesha KR. HPLC‑UV method for simultaneous
determination of sparfloxacin and dexamethasone sodium
Conflicts of interest phosphate in eye drops. Pak J Pharm Sci 2019;32:1057‑61.
There are no conflicts of interest.
12. Duarah S, Sharma M, Wen J. Rapid and simultaneous determination
of dexamethasone and dexamethasone sodium phosphate using
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