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VALIDATIÓN OF ANALYTICAL METHOD BY UV

SPECTROPHOTOMETRIC QUANTIFICATION OF GEMFIBROZIL

Herrera et. al., 137–150

VALIDATION OF ANALYTICAL METHOD

BY UV SPECTROPHOTOMETRIC

QUANTIFICATION OF GEMFIBROZIL

INCORPORATED IN THE MICROEMULSIONS

María Herrera P.

1

, José Cantos C.

1

, Kevin Muñoz S.

1

, Jovan Durán A.

2

,

Julio Vinueza G.

3

& Fernanda Kolenyak-S.

1*

Recibido: 23 de octubre 2020 / Aceptado: 14 de diciembre 2020

DOI: 10.26807/ia.v9i1.195

Keywords: Analytical Validation, Gemfibrozil, Microemulsions,

UV spectrophotometric

ABSTRACT

The present study aims to validate an analytical method by UV spectrophoto-

metric for the quantification of gemfibrozil incorporated into microemulsions.

Experimental development by constructing a ternary phase diagram for the iden-

tification of microemulsions. The characterization of the microemulsions was

1 Universidad de Guayaquil, Facultad de Ciencias Químicas, Guayaquil, Ecuador. (mariaelizabeth-

herreraparedes@gmail.com; josecantossc@gmail.com; kevinjoelmuoz25@hotmail.es; *correspon-

dência: fernandakolenyak@outlook.com)

2 Universidade Estadual Paulista (UNESP), Instituto de Química, Departamento de Química Analí-

tica, Araraquara, SP, Brasil (alonso.jdr@gmail.com;)

3 Pontificia Universidad Católica del Ecuador, Facultad de Ciencias Exactas y Naturales, Escuela de

Química, Quito, Ecuador (julio_vinueza@hotmail.com)

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carried out by the polarized light microscopy technique. The analytical method

was validated according to the parameters described by the United State Phar-

macopeia (USP) 39 NF34 and International Conference on Harmonization

(ICH) (2005), which evaluated linearity, robustness, intra and inter day accuracy,

specificity accuracy, limits of detection and quantification. The developed met-

hod demons trated linearity with a coefficient of determination (R2) of 0.9898,

in addition to precision, accuracy and robustness for each of the tests with va-

lues that presented a coefficient of variation of less than 5 %. The results obtai-

ned were favorable, since they comply with the parameters stipulated by USP

39-NF34 (2016) and ICH (2005), indicating that the method is effective for the

intended purpose.

INTRODUCTION

The prolonged drug release systems

are classified as new technological

alternatives used to control the con-

centration and time of drug release

after administration. These systems

can be solid dispersions, micro and

nanoparticles, liposomes, microe-

mulsions etc. (Huo et al., 2017; Da-

voodi et al., 2018; El Maghraby,

Arafa, & Essa, 2018).

Microemulsions (MEs), are systems

composed of water and oil that are

stabilized by surfactants, their main

characteristic is thermodynamic sta-

bility, due a large amount of energy

is not required for their formation.

Unlike emulsions, microemulsions

have transparent aspects, this is It is

due to the small size of the particles,

which can reach 500 nm. ME can be

characterized as oil in water (O/W)

and water in oil (W/O), which con-

fers the particularity of being able to

incorporate lipophilic and hydrophi-

lic drugs (Callender et al., 2017; Fe-

rreira et al., 2018; Gvaramia et al.,

2018).

Gemfibrozil (GFZ) is a drug that acts

as a cholesterol reducer, derived from

fabric acid and chemically known as

5- (2,5-dimethylphenoxy) -2,2-dime-

thylpentanoic acid. Its prescription is

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also made in order to reduce the risk

of stroke, heart attack or other cardiac

complications as it increases high

density lipoproteins (LDL) (Tornio et

al., 2017). Despite having studied the

interactions, it is not yet possible to

accurately quantify the drug within

the systems matrix due to the lack of

validated methods for this purpose.

The validation of a methodology is

important as it aims to demonstrate

the suitability of the method to carry

out an analysis. The methods of

analysis used in the quality control of

pharmaceutical products must have

been validated prior to routine use.

The validation of a test method is in-

tended to demonstrate the suitability

of the method to carry out an analysis

(USP 39 NF 34 2016; ICH, 2005;

Cáñez & García, 2015).

Ultraviolet-Visible Spectroscopy (UV-

Vis) is a technique that is based on

the ability of certain molecules to ab-

sorb radiation such as those corres-

ponding to the visible UV spectrum.

The use of a spectrophotometer

allows readings in a wavelength

range of 190 to 800 nm, simulta-

neously considering factors such as

atomic structure, medium conditions

(pH, temperature, ionic strength, die-

lectric constant) which finally makes

the information provided of the

energy states, atomic or molecular, of

the study sample allow its determina-

tion and characterization. The UV-

spectrophotometry is an inexpensive,

easy, reliable, fast and useful, meets

the requirements of quality control of

pharmaceutical and cosmetic indus-

tries. As an alternative to the existing

method by HPLC (Valcárcel, Gonzá-

les y Valcárcel, 2008; Cubas et al.,

2018; Maldonado et al., 2018). The

aim of this work is to validate an

analytical method to gemfibrozil in-

corporated in the microemulsions.

MATERIALS AND METHODS

Materials

Sodium Chloride (NaCl), Potassium

Chloride (KCl), Calcium Chloride

(CaCl

2

), Sodium Phosphate (NaHPO

4

),

Glucose (C

6

H

12

O

6

), Sodium Hydro-

xide (NaOH), Gemfibrozil were pur-

chased from Sigma-Aldrich CO (USA).

Methods

Phase diagram construction

The ternary phase diagram was pre-

pared using tween80, oleic acid and

Milli Q water at 25 °C. In the mixture

of water/oil was added the surfactant

until the solution is completely trans-

parent, which was indicative of the

formation of the single phase. Once

the candidate sample was separated,

the sample was prepared with and

without drug for the validation of the

analytical methodology.

Polarized light microscopy

The microemulsion structure was

analyzed by means of a photomicros-

cope (Leica DMLP microscope)

equipped with a camera: firstly,

under the bright field and secondly,

under the polarized light (using cross-

polarizers) in order 5 to investigate

the presence of the liquid crystalline

phase in each sample. Leica IM 1000

software was used to analyze the ob-

tained micrographs.

Preparation of buffer TC199 with

glucose at pH 7.4

The buffer was prepared according

described by Silva et al (2008). Briefly

145 mM NaCl; 4.56 mM KCl; 1.25

mM CaCl

2

2H

2

O; 5 mM NaHPO

4

were weighed and leading to a final

volume of 1000 mL of Milli Q water,

the glucose was added, and the solu-

tion was adjusted to a pH of 7.4 using

1N NaOH.

Analytical method validation

The methodology was performed

using the parameters defined by USP

39 NF 34 (2016) and ICH Q2 (2005).

All analyzes were performed at a wa-

velength of 276 nm, this value refers

to the maximum absorption wave-

length of GFZ (USP 38, 2015).

Linearity

The linearity of the method was de-

termined by obtaining three analyti-

cal curve of gemfibrozil/microemul

sion (GFZ/MEs) in absolute ethanol

(250 mg/mL). Transferred aliquots of

stock solution to a volumetric flask to

obtain nine concentrations, from

0.02 to 0.09 mg/mL of GFZ and the

volume was completed with absolute

ethanol, then the absorbance-absor-

bance of the samples were measured

in 276 nm using blank ethanol. The

dilutions were prepared in triplicate.

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Precision

The data were analyzed at a wave-

length 276 nm. The precision of the

analytical curves was determined in

three consecutive days for intra- and

inter-day.

Accuracy

The accuracy is the closeness of the

results obtained by the method under

study in relation to the true value for

the quantitative analysis (ICH 2005).

The accuracy was determined by tes-

ting the recovery of GFZ in ethanol,

which was calculate the percentage of

recovery of known amount of drug

added to the sample using Equation 1.

%Recovery: Y/X x100 % (1)

Y = absorbance of the sample; X =

standard absorbance; acceptance cri-

teria for recovery: 98-102 %.

Limit of detection (LOD), limit of

quantitation (LOQ)

To determine the LOD and LOQ,

three solutions prepared in triplicate,

near the lower limit of the analytical

curve. The LOD and LOQ were cal-

culated based on the equations (2

and 3) as describe in ICH (2005), it is

expressed as:

LOD = 3(S.D./a) (2)

LOQ = 10(S.D./a) (3)

where S.D of y-intercepts and S is the

slope of the calibration curve.

Robustness

The robustness should show the relia-

bility of an analysis related to delibe-

rate variations in method parameters

ICH (2005). The robustness was de-

termined by analyzing the sample

under the variation of the pH and

manufacture of ethanol.

Specificity

The specificity was carried out under

two standards: gemfibrozil and mi-

croemulsion without the drug, to de-

termine that there would be no

interference from the excipients pre-

sent in the formulation.

Encapsulation efficiency

The entrapment efficiency (EE) was

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determined to access the extent of

GFZ incorporation in the microemul-

sion. EE of GFZ-loaded MEs was de-

termined by measuring the concen-

tration of the free unloaded GFZ in the

aqueous medium of the MEs suspen-

sion. Briefly, one gram of GFZ-loaded

MEs was weighted and ultracentrifu-

ged for 20 min at 15000 rpm, appro-

ximately. Then, the amount of GFZ in

the supernatant was determined by

UV spectrophotometric analysis. The

entrapment efficiency (EE) was calcu-

lated by the equation 4.

RESULTS

Phase diagram construction

In the present study a ternary phase

diagram was construed and microe-

mulsion region was found using sur-

factant concentration rate between

10-40 %. The Figure 1 exhibit the

phase diagram and the photograph of

the selected microemulsion.

Figure 1. The photograph of selected

microemulsion

Polarized light microscopy

The Figure 2 exhibit eh light micros-

copy to microemulsion containing

GFZ

Figure 2. Light microscopy to

microemulsion containing GFZ

EE (%) = (4)

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Validation of analytical method

Lineality

In order to obtain the linearity results,

a calibrated analytical curve is cons-

tructed, so that the dilutions are ela-

borated from a parent solution, the

value obtained from R

2

is 0.9984 as

shown in Figure 3 and the standard

deviation values.

Figure 3. The linearity results

The standard deviation (SD) and coef-

ficient of variation (CV) data is shown

in Table 1.

Precision

This parameter was assessed by ap-

plying the intra and inter day tests

through the analysis of 3 samples of

different concentrations, the mini-

mum (0.02 mg/mL), the medium

(0.05 mg/mL) and the maximum (0.09

mg/mL) as showed in the Table 2.

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Accuracy

The accuracy of the proposed met-

hod was assessed by determining the

average recoveries of samples using

the standard addition method. The

Table 3 show the results of accuracy.

Table 1. Standard deviation and coefficient

of variation of the curve data

Concentration Average DS (±) CV

(mg/mL) %

0.09 0.6152 0.0102 1.66

0.08 0.5492 0.0100 1.82

0.07 0.4651 0.0027 0.58

0.06 0.3964 0.0028 0.72

0.05 0.3265 0.0042 1.29

0.04 0.2662 0.0045 1.69

0.03 0.1950 0.0035 1.79

0.02 0.1358 0.0022 1.61

Table 2. Standard deviation and coefficient of variation

of the Inter and Intraday precision

Interday Intraday

Concentration Average SD (±) CV Concentration Average SD (±) CV

(mg/mL) (%) (mg/mL) (%)

0.09 0.5963 0.0029 0.48 0.09 0.6009 0.0006 0.10

0.05 0.3262 0.0004 0.13 0.05 0,3263 0,0002 0,07

0.02 0.1346 0.0004 0.27 0.02 0.1358 0.0005 0.37

Table 3. Accuracy test results

Concentration Average SD (±) CV % Recovery

(mg/mL) (%)

0.09 0.5968 0.0018 0.30 101.15%

0.05 0.3389 0.0014 0.41 101.16%

0.02 0.1350 0.0005 0.39 99.11%

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Specificity

The specificity of the method was

evaluated under 2 standards gemfi-

brozil and microemulsion without

drug, in order to determine that there

was no interference from the exci-

pients present in the formulation as

shown in Figure 4.

Figure 4. Specificity of microemulsion without gemfibrozil

Robustness

The robustness test was realized mo-

difying the manufacture and the pH

of the buffer solution. These parame-

ters were selected due the ease ac-

cess. The results of the selected varia-

bles in the robustness assessment are

shown in Table 4.

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LOD and LOQ

The detection limit is the lowest

amount of the analyte that can be de-

tected but do not necessarily quanti-

fied, while the limit of quantification

is the lowest amount of the analyte

that can be determined with accu-

racy and precision ICH (2005). The

LOD and LOQ values were 0.062

and 0.189 mg/mL respectively, this

results indicate the excellent sensiti-

vity of the analytical method.

Encapsulation efficiency

The encapsulation efficiency to GFZ

was 88 % at the MEs. The results for

both NLCs analyzed are close to

those described by Addas et al.

(2019) and were satisfactory, since

they show high entrapment efficiency

in the matrix of MEs.

Table 4. Robustness test results

Concentration

0.05mg/mL

Buffer pH 7.0 Buffer pH 7.4

Average SD (±) CV % Average SD (±) CV %

Manufacture 1 0.3267 0.0001 0.018 0.3263 0.0003 0.08

0.3264 0.0002 0.047 0.3264 0.0002 0.047

0.3264 0.0002 0.071 0.3263 0.0002 0.064

Manufacture 2 0.3262 0.0001 0.018 0.3265 0.0001 0.018

0.3260 0.0002 0.047 0.3263 0.0002 0.064

0.3259 0.0002 0.0469 0.3264 0.0001 0.0177

DISCUSSION

In the present work, we have valida-

ted a method to quantification of

gemfibrozil incorporated in the mi-

croemuslions. It is possible to ob-

serve a transparent solution with a

single phase which indicates the for-

mation of microemulsion. It is sug-

gested due the spontaneous forma-

tion and thermodynamic stability,

and a dark field was observed in the

polarized microscopy which suggest

the microemulsion formation. It was

possible to observe the presence of a

black field, which can suggest mi-

croemulsions formation. According

to Froelich et al. (2017), a main fea-

ture of these microemulsion systems

is dark field observation.

To validation method, it was demons-

trates the linearity expressed by the

relationship between the concentra-

tion of gemfibrozil present obtaining

a value of R

2

0.9984, indicating that

the curve is linear for the intended

purpose. Similar results were found

by Gaete (2014), the author validated

an analytical methodology for the ap-

plication of paracetamol in a bio-

exemption study, and obtained a

value of 0.999 in the R

2

, which indi-

cated that the curve is linear for the

purpose of the work.

The acceptance of linearity will de-

pend on the value obtained in the co-

rrelation coefficient and the intersec-

tion of the line are its axis. This value

indicates the linear regression of the

concentration against absorbance.

The correlation coefficient value

equal to or greater than 0.99 is nor-

mally acceptable, since it indicates

that the analytical curve is linear USP

39 NF 34 (2016). In this context, the

analytical curve is considered as a re-

ference for the quantification of gem-

fibrozil.

The data presented in Table 2 showed

that the method is precise for the

quantification of gemfibrozil incorpo-

rated at the microemulsion. The coef-

ficient of variation values was below

0.5 %, which indicate that the met-

hod is sufficiently precise. The results

are in accordance with the provisions

of the ICH (2005), which suggests

that the coefficient of variation for

this parameter should be less than 3.0

%. Cáceres, et al (2016) obtained a

coefficient of variation of less than 1

%, with the results presented, both

authors argue that the method is ac-

curate. With the above it is shown

that the method is accurate for the in-

tended purpose.

The results presented in the Table 3

are in accordance with the official

validation regulations. The ICH

(2005) stipulates that the recovery

percentage must be in a range of 80

to 120 %. Lozano et al., (2018) vali-

dated an analytical method for the

quantification of alprazolam in ta-

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blets, in the precise parameter, the re-

sults obtained a recovery of 100.69

%, in this way, the authors indicated

that the parameter is within the limits

stipulated by ICH (2005) and USP 39

NF34 (2016).

The specificity of the drug could be

determined by a wavelength scan,

where we can show that the highest

reading peak corresponds to the 276

nm suggested by USP 39 NF34

(2016) as the wavelength for GFZ. In

addition, it can be evidenced that no

interference is generated between ME

without drug and the sample with

GFZ demonstrating that microemul-

sion excipients do not interfere with

gemfibrozil reading.

CONCLUSION

In the present project, a method pro-

posed an analytical derivative UV

spectrophotometric was developed.

This method showed advantages re-

lated to the simplicity, fastness and

low-cost conditions. All validation

parameters were satisfactory, the re-

sults showed that the method is sui-

table for quantification of gemfibrozil

incorporated in microemulsion, as

consistent with the requirements of

ICH, thus providing reliability to the

analyses proposed.

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