Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2013 – vol. 117, no. 4
PHARMACY
ORIGINAL PAPERS
DEVELOPMENT AND VALIDATION OF A SPECTROPHOTOMETRIC METHOD
FOR QUANTITATIVE DETERMINATION OF NEW PRESERVATIVES FROM
PHARMACEUTICAL FORMS
Catalina Daniela Stan1, Alina Ştefanache 2, Maria Drăgan 1, Andreea Maria Corciovă 3
University of Medicine and Pharmacy “Grigore T. Popa”- Iaşi
Faculty of Pharmacy
1. Drug Industry and Pharmaceutical Biotechnology Department
2. General and Innorganic Chemistry Department
3. Drug Control Department
DEVELOPMENT AND VALIDATION OF A SPECTROPHOTOMETRIC METHOD FOR
QUANTITATIVE DETERMINATION OF NEW PRESERVATIVES FROM PHARMACEUTICAL FORMS (Abstract): Aim: A spectrophotometric UV-VIS fast, sensitive and simple
method was developed for quantitative determination of two new antimicrobial preservatives
(oxi-acetyl-mandelic acid and oxi-propionyl mandelic acid) and validated according to validation quides. Material and methods: The absorption spectra were recorded at 200–400 nm, and
the absorbances were measured at 256 nm for oxi-acetyl mandelic acid and 258 nm for oxipropionyl mandelic acid. Optimum working conditions were established and the method was
validated. The developed and validated method was applied to determine these two preservatives from ointments, in which the preservatives were incorporated. Results: The method has a
good linearity in the concentration range 0.1–1.5 mg/ml (for oxi-acetyl mandelic acid) and 0.1–
1 mg/ml (for oxi-propionyl mandelic acid). The RSD for the precision of the method was
0.96328 for oxi-acetyl mandelic acid and 0.797612 for oxi-propionyl mandelic acid. The relative standard deviation was 0.726367% for oxi-acetyl mandelic acid and 0.726234% for oxipropionyl mandelic acid. In the ointment samples, the concentration of oxi-acetyl mandelic acid and oxi-propionyl mandelic acid were within the limits specified by pharmacopoeia for
ointments. Conclusions: The developed and validated spectrophotometric method, for the determination of oxi-acetyl mandelic acid and oxi-propionyl mandelic acid is simple, easy to perform, fast and cost effective. Keywords: VALIDATION, OINTMENTS, PRESERVATIVES,
SPECTROPHOTOMETRIC METHOD, MANDELIC ACID
Mandelic acid is known as an antiseptic,
being active against some pathogens
(Staphylococcus aureus, E. coli, Proteus
sp., Aerobacter sp. etc.). It is used in urinary infections (oral use), or dermic infections (inflammatory acne), for external use.
Starting from this acid, we synthesized
some mandelic acid derivatives, two of
them (oxi-acetyl mandelic acid and oxi-
1014
propionyl mandelic acid) presenting high
antimicrobial activity compared to mandelic acid.
For these two derivatives, oxi-acetyl
mandelic acid and oxi-propionyl mandelic
acid, were carry out some studies: there
were confirmed their chemical structures
(by elemental analysis, UV-VIS, IR, RMN
1
H spectral analysis), there were estab-
Development and validation of a spectrophotometric method for quantitative determination
of new preservatives from pharmaceutical forms
lished the minimum inhibitory concentrations (MCI=125 µg/ml for oxi-acetyl mandelic acid and MCI=500 µg/ml for oxipropionyl mandelic acid) and lethal dose
50%, after oral administration (LD50 =800
mg/kg for oxi-acetyl mandelic acid and
LD50=3000 mg/kg for oxi-propionyl mandelic acid) (1, 2).
This paper presents the development of
a fast and sensitive spectrophotometric
UV-VIS method for oxi-acetyl mandelic
acid and oxi-propionyl mandelic acid determination. The spectrophotometric method was validated according to validation
quides and applied to oxi-acetyl mandelic
acid and oxi-propionyl mandelic acid determination from pharmaceutical forms
(ointments) (3, 4, 5).
MATERIAL AND METHODS
Apparatus:
- Jasco V530 spectrophotometer, quartz
cells (1=1 cm);
- IKA-Werke basic ultrasonic bath.
Materials and reagents:
 oxi-acetyl mandelic acid, new synthesized preservative, obtained in faculty lab,
for which we have validated the structure
and established the MCI, the results have
been previously published in journals;
 oxi-propionyl mandelic acid, new synthesized preservative, obtained in faculty
lab, for which we have validated the structure and established the MCI, the results
Rp/
Cetyl alcohol
5g
Sunflower seed oil
20 g
Lanolin
5g
Vaseline
20 g
Oxi-acetyl mandelic acid 0.0125 g
Distilled water q.s. ad 100 g
have been previously published in journals;
 absolute ethanol (p.a.) (Merck KGaA);
 cetyl alcohol, lanolin, vaseline, sunflower seed oil were produced by Redox,
Bucureşti, România.
There were prepared oxi-acetyl mandelic acid and oxi-propionyl mandelic acid
stock solutions and then were obtained
suitable dilutions in absolute ethanol. Taking into account that those two preservatives are active at low concentrations, the
determination was performed at concentrations close to the active ones. The absorption spectras were recorded between 200–
400 nm, and the absorbances were measured at 256 nm for oxi-acetyl mandelic acid
and 258 nm for oxi-propionyl mandelic
acid in a 1 cm cell versus a blank of absolute ethanol.
The developed method was validated by
studying the following parameters: linearity, detection limit, quantification limit,
precision, in accordance with the bioanalytical method validation guidelines (3, 4, 5).
The developed and validated method
was applied to determine these two preservatives from pharmaceutical forms
(ointments). According to MCI`s, the preservatives were embedded in ointments in
0.0125 g% concentration for oxi-acetyl
mandelic acid and in 0.05 g% concentration for oxi-propionyl mandelic acid. The
ointments were prepared by the formula
presented below:
Rp/
Cetyl alcohol
5g
Sunflower seed oil
20 g
Lanolin
5g
Vaseline
20 g
Oxi-propionyl mandelic acid 0.05 g
Distilled water
q.s. ad
100 g
1015
Catalina Daniela Stan et al.
These two preservatives were extracted
from ointments with gently heated absolute
ethanol and, after filtration; the samples
were spectrophotometric analyzed at 256
nm for oxi-acetyl mandelic acid and at 258
nm for oxi-propionyl mandelic acid, respectively.
RESULTS AND DISCUSSION
The method was validated by studying
the following parameters: linearity, detection limit, and quantification limit, preci-
No.
1
2
3
4
5
6
7
8
No.
1
2
3
4
5
6
7
8
TABLE I
Absorbance values for establish the linearity of the method
Absorbance (λ=256 nm)
Oxi-acetyl mandelic acid
concentration mg/ml
I
II
III
0.1
0.0375
0.0322 0.0437
0.3
0.1675
0.1632 0.1598
0.5
0.3331
0.3129 0.3290
0.7
0.4589
0.4600 0.4656
0.9
0.5999
0.6231 0.6202
1.1
0.7325
0.7833 0.8179
1.3
0.9128
0.9323 0.9311
1.5
1.1109
0.9834 1.1991
Absorbance (λ =258 nm)
Oxi-propionyl mandelic acid concentration mg/ml
I
II
III
0.1
0.1222
0.0982 0.1101
0.3
0.2835
0.301 0.2989
0.4
0.3899
0.3943 0.4101
0.5
0.4952
0.4888 0.4903
0.6
0.5921
0.5899 0.5932
0.7
0.6854
0.7128 0.7001
0.8
0.8201
0.7967 0.8024
1
1.0098
1.0156 1.0167
The average values of absorbance are
graphically represented according to concentrations (fig. 1 and fig. 2).
Statistical analysis of experimental data
leads to regression line equations, presented in table II. There were calculated the
standard error of regression curve (SE), the
1016
sion.
Linearity. There were prepared stock
solutions of oxi-acetyl mandelic acid, respectively oxi-propionyl mandelic acid,
from which were obtained the working
solutions in a concentration range of 0.1–
1.5 mg/ml (for oxi-acetyl mandelic acid)
and 0.1–1 mg/ml (for oxi-propionyl mandelic acid). For each concentration, three
determinations were performed and the
average value of absorbance was calculated
for each range (tab. I).
Average
0.0378
0.1635
0.3235
0.4615
0.6144
0.7779
0.9254
1.0978
Average
0.1102
0.2944
0.3981
0.4914
0.5917
0.6994
0.8064
1.0140
Person correlation coefficient (r 2 ), the detection limit (LOD) and the quantification
limit (LOQ). In tab. III are prezented the
statistical data.
In conformity with the obtained data,
the developed method was linear in the
range of the studied concentration.
Development and validation of a spectrophotometric method for quantitative determination
of new preservatives from pharmaceutical forms
Fig. 1. Regression line for oxi-acetyl
mandelic acid determination
Fig. 2. Regression line for oxi-propionyl
mandelic acid determination
TABLE II
Regression line equations for the two preservatives determination
Substance
Regression line equations
Oxi-acetyl mandelic acid
Absorbance = 0.75867x - 0.05671
Oxi-propionyl mandelic acid
Absorbance = 1.00851x - 0.00396
TABLE III
Statistical data regarding the two preservatives determination
Oxi-acetyl
Oxi-propionyl
mandelic acid
mandelic acid
2
0.99897
0.99920
Person coefficient (r )
0.012876
0.008863
Standard error
Intercept
- 0.05671
- 0.00396
Slope
Detection limit (LOD) mg/ml
Quantification limit (LOQ) mg/ml
0.75867
0.05
0.17
1.00851
0.03
0.08
Precision of the system and method. Precision of the system was studied for the 1.3
mg/ml concentration of oxi-acetyl mandelic
acid and 0.6 mg/ml concentration of oxipropionyl mandelic acid, for 10 assays performed in the same conditions. The results
and the statistical analysis for standard deviation (SD) and relative standard deviation
(RSD %) are presented in table IV.
Processing data lead to a relative standard deviation RSD=0.96328% for oxiacetyl mandelic acid and RSD=0.797612%
for oxi-propionyl mandelic acid, compared
to the maximum of 2% proposed by the
European standards, so the systems are
considered to be precise.
1017
Catalina Daniela Stan et al.
TABLE IV
System precision
Oxi-acetyl
Oxi-propionyl
mandelic acid
mandelic acid
Absorbance
Absorbance
1
0.9234
0.5917
2
0.9231
0.5998
3
0.9111
0.6001
4
0.9323
0.6013
5
0.9101
0.5928
6
0.9321
0.6032
7
0.9278
0.5925
8
0.9109
0.5922
9
0.9215
0.5912
10
0.9311
0.5922
Average
0.92234
0.5957
Standard Deviation (SD)
0.008885
0.004751
Relative standard deviation (RSD) %
0.96328
0.797612
Det. no.
The precision of the method was investigated in a range of 20% compared to interest
concentration, grouped on a number of 3
assays in 3 concentrations levels. Using the
regression line equation the concentrations
and recovery were calculated (tab. V).
From statistical analysis we obtained a
relative
standard
deviation
RSD=
0.726367% for oxi-acetyl mandelic acid
and RSD=0.726234% for oxi-propionyl
mandelic acid, compared to the maximum
of 5% proposed by the european standards,
so the proposed method is precise.
The analysis of the two preservatives
1018
from ointments. The presence of the oxiacetyl mandelic acid and oxi-propionyl
mandelic acid was carried out by comparing the spectras obtained after extraction
with the standards ones. The obtained data,
after the two preservatives quantification
from the ointments were compared with the
limits recommended by pharmacopoeia
(tab. VI) (6, 7).
The experimental data shows us that the
concentrations of oxi-acetyl mandelic acid
and oxi-propionyl mandelic acid from the
studied ointments are in the recommended
ranges for ointments in pharmacopoeia (6, 7).
Development and validation of a spectrophotometric method for quantitative determination
of new preservatives from pharmaceutical forms
TABLE V
The calculated concentrations and recovery for the two preservatives
0.7901
1.11
100.90
0.7821
1.10
100,00
0.9312
1,30
100.00
0.9265
1.29
99.23
0.9335
1.31
1.0801
Recovery (%)
99.09
Calculated
conc.
1.09
Theoretic
conc. mg/ml
0.7729
Absorbance
1.5
Recovery (%)
1.3
Calculated
conc.
1.1
Oxi-propionyl mandelic acid
Absorbance
Theoretic
conc. mg/ml
Oxi-acetyl mandelic acid
0.3998
0.4003
100.07
0.4049
0.4054
101.35
0.4001
0.4006
100.15
0.5965
0.5953
99.23
0.6015
0.6003
100.05
100.76
0.5996
0.5984
99.74
1.49
99.33
0.8024
0.7995
99.94
1.0930
1.51
100.66
0.7998
0.7969
99.62
1.0850
1.50
100.00
0.8112
0.8082
101.35
Average
99.99
Average
100.16
SD
0.727444
RSD %
0.726234
Statistical data
SD
0.72634
RSD %
0.726367
0.4
0.6
0.8
Statistical
data
TABLE VI
The calculated concentrations and recovery
for the two preservatives determination from ointments
Theoretical conc.
± admissible
deviation FRX
Det.
no.
1.
2.
Oxi-acetyl
mandelic acid
0.0125 ± 7.5%
3.
1.
2.
Oxi-propionyl
mandelic acid
0.05 ± 7.5%
3.
CONCLUSIONS
A simple, fast and sensitive UV-VIS
spectrophotometric method was developed
Calculated average conc.
± standard deviation
Recovery
%
0.01251 ± 0.0001
100.08
0.01249 ± 0.0001
99.92
0.01232 ± 0.0001
98.56
0.0508 ± 0.0501
101.60
0.0497 ± 0.0501
99.40
0.0498 ± 0.0501
99.60
and validated for the assay of two new
preservatives (oxi-acetyl mandelic acid and
oxi-propionyl mandelic acid). The valida-
1019
Catalina Daniela Stan et al.
tion was performed according to validation
guides. The developed and validated spectrophotometric method was applied to determine these two preservatives from
pharmaceutical forms (ointments). The
mean recovery in the ointments of the two
preservatives was in the range of 98.56–
101.60%.
REFERENCES
1. Stan C, Nastase V, Pavelescu M et al. Sinteza unor noi derivaţi ai acidului mandelic cu acţiune conservantă.Sinteza şi studiul toxicităţii acute. Rev Med Chir Soc Med Nat 2004; 108(1): 185-188.
2. Stan C, Poiată A, Dumitrache M, Nastase V. Minimum inhibitory concentration determination for
new synthesis preservatives, esters of DL-mandelic acid-Note II. Eur J Drug Metab Pharmacokinet
2003; 28(1): 39.
3. Rouessac F, Rouessac A. Chemical analysis: Modern instrumental methods and techniques. Chichester: John Willey & Sons Ltd., 2000.
4. Oprean R, Rozet E et al. Ghid de validare a procedurilor analitice cantitative. Cluj Napoca: Editura
Medicală Universitară ”Iuliu Haţieganu”, 2007.
5. Roman L, Bojiţă M, Săndulescu R, Muntean DL. Validarea metodelor analitice. Bucureşti: Editura
Medicală, 2007.
6. *** Farmacopeea Română. Ediţia a X-a. Bucureşti: Editura Medicală, 1993.
7. *** European Pharmacopoeia. 7.0 edition, on line, 2011.
NEWS
RAMAN SPECTROSCOPY FOR DIFFERENTIATION OF CAUSATIVE AGENTS
OF ONYCHOMYCOSIS
The discriminative power of Raman spectroscopy for differentiation of important dermat ophytic and non-dermatophytic agents of onychomycosis in human nail was analyzed in a
study by Smijs et al. An ex vivo nail model was used to determine the capacity of Raman
spectroscopy to distinguish between Trichophyton rubrum, T. mentagrophytes, T. tonsurans,
Scopulariopsis brevicaulis and Candida albicans. For this purpose, Raman spectra
(200/sample) were taken from 50-μm slices of infected nail using a 2500 High-Performance
Raman Module and 785-nm diode laser. Analysis of spectra showed differences between the
dermatophytes (with T. rubrum being most different) and between dermatophytes the nondermatophytes S. brevicaulis and C. albicans. In conclusion, Raman spectroscopy allows for
specific and non-invasive differentiation of causative organisms of onychomycosis (Smijs
TG, Jachtenberg JW, Pavel S, Bakker-Schut TC et al. Detection and differentiation of causative organisms of onychomycosis in an ex vivo nail model by means of Raman spectroscopy.
J Eur Acad Dermatol Venereol. 2013. doi: 10.1111/jdv.12324).
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