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Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917

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Journal of Pharmaceutical and Biomedical Analysis


journal homepage: www.elsevier.com/locate/jpba

Development and validation of a reversed phase liquid chromatographic method


for analysis of griseofulvin and impurities
Getu Kahsay a , Aremu Olajire Adegoke b , Ann Van Schepdael a , Erwin Adams a,
a
Laboratorium voor Farmaceutische Analyse, Faculteit Farmaceutische Wetenschappen, KU Leuven, O&N2, PB-923, Herestraat 49, B-3000 Leuven, Belgium
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ibadan, Orita UI Ibadan, Nigeria

a r t i c l e i n f o a b s t r a c t

Article history: A simple and robust reversed phase liquid chromatographic method was developed and validated for the
Received 5 February 2013 quantitative determination of griseofulvin (GF) and its impurities in drug substances and drug products
Accepted 23 February 2013 (tablets). Chromatographic separation was achieved on a Discovery C18 (250 mm 4.6 mm, 5 m) column
Available online 4 March 2013
kept at 30 C. The mobile phase consisted of a gradient mixture of mobile phase A (water0.1% formic
acid pH 4.5, 80:20, v/v) and B (ACNwater0.1% formic acid pH 4.5, 65:15:20, v/v/v) pumped at a ow
Keywords:
rate of 1.0 mL/min. UV detection was performed at 290 nm. The method was validated for its robustness,
Griseofulvin
sensitivity, precision, accuracy and linearity based on ICH guidelines. The robustness study was performed
Impurities
Analysis
by means of an experimental design and multivariate analysis. Satisfactory results were obtained from
MS/MS the validation studies. The use of volatile mobile phases allowed for the identication of three main
Liquid chromatography impurities present above the identication threshold using mass spectrometry (MS). The developed LC
method has been applied for the assay and impurity determination of GF drug substances and tablets.
The method could be very useful for the quality control of GF and its impurities in bulk and formulated
dosage forms.
2013 Elsevier B.V. All rights reserved.

1. Introduction In the United States Pharmacopeia, both the drug substance


and the dosage forms are assayed by LC using a stationary phase
Griseofulvin (GF) was the rst orally available antimycotic agent with nitrile groups bonded to porous silica particles [23]. How-
which has been in use for over forty years [1]. The drug is still rele- ever, there is no specication for related substances. The European
vant due to its reasonable price, what is certainly advantageous in Pharmacopoeia [2] describes a GC method for the determination
developing countries. GF is produced by Penicillium griseofulvum or of dechlorogriseofulvin and dehydrogriseofulvin while the assay of
obtained by other means [2]. Related substances of GF were iden- the drug substance is performed by UV spectroscopy. The Interna-
tied from fermentation media and include dechlorogriseofulvin, tional Pharmacopoeia [24,25] species a UV method for assay of GF
dehydrogriseofulvin, dihydrogriseofulvin, tetrahydrogriseofulvin, without any method for related substances.
griseofulvic acid and isogriseofulvin [3]. The rst two can also be In literature, only a few methods have been reported for the
formed by degradation. The chemical structures of griseofulvin and analysis of GF in the drug substance or dosage forms. Some of
its related impurities are presented in Fig. 1. the methods are rather old like the one of Holbrook et al. [26]
The analysis of GF has been focused mainly on its determina- who described in 1963 a chromatographic method for the assay
tion in biological uids such as plasma, serum and urine. These of GF in the presence of several related substances employing
methods include thin layer chromatography (TLC) [46], LC [711] Celite and measuring the eluate fractions by UV. In 1980, Town-
and gas chromatography (GC) [6,1214]. Recently, an electrospray ley and Roden [27] published an LC method using a Zorbax CN
ionization LCMS/MS method was validated and adopted for the column to determine the purity of GF bulk drug substances, to
determination of GF in human plasma [15]. Characterization and assay GF in powders, tablets, capsules and to separate GF from its
determination of GF in some other sample matrices such as moulds, metabolites. Thoma and Kbler [28] carried out photodegradation
marine strains, food and feeds and bioavailability studies have also of antimycotic drugs, ucytosine and griseofulvin and noted that
been reported [1622]. dechlorogriseofulvin was formed in aqueous solutions as well as in
ethanolic and methanolic solutions. The authors mentioned that in
spite of the photo-instability of GF, no pharmacopoeia recommends
storage protected from light.
Corresponding author. Tel.: +32 16323444; fax: +32 16323448. As no up-to-date LC method is available at this moment for
E-mail address: Erwin.Adams@pharm.kuleuven.be (E. Adams). the determination of GF and its impurities, the development and

0731-7085/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2013.02.035
10 G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917

2.3. Instrumentation and liquid chromatographic conditions

2.3.1. Chromatography
The LC system from Dionex Softron GmbH (Germering,
Germany) was equipped with a high pressure pump (P680ALPG),
autosampler (ASI-100T) and UV/VIS detector (UVD170U). For data
processing and acquisition, Chromeleon software version 6.80 from
Dionex (Sunnyvale, CA, USA) was used. An ultrasonicator from
Branson Ultrasonics Corporation (Danbury, CT, USA) and a pH metre
from Metrohm (Herisau, Switzerland) were used to dissolve the
sample and measure the pH of the mobile phase, respectively.
The column was kept in a water bath at a temperature of 30 C
using a Julabo EM immersion thermostat (Seelbach, Germany).
Final chromatographic separations were achieved on a Discov-
ery C18 (250 mm 4.6 mm, 5 m) column obtained from SUPELCO
(Bellefonte, PA, USA). The mobile phase was a gradient mixture of
mobile phase A (water0.1% formic acid pH 4.5, 80:20, v/v) and B
(ACNwater0.1% formic acid pH 4.5, 65:15:20, v/v/v) pumped at a
ow rate of 1.0 mL/min. The gradient programme [time (min)/%B]
was set as 0/50, 3/50 to 13/60 to 16/90, 20/90 to 24/50, 30/50. The
gradient dwell volume of the system is 1.6 mL. The injection vol-
ume was 10 L and UV detection was performed at 290 nm. The
mobile phases were degassed by sparging with helium.

2.3.2. Mass spectrometry


An LCQ ion trap mass spectrometer from ThermoFinnigan (San
Jose, CA, USA) equipped with an electrospray ionization source (ESI)
interface operated in the positive ion mode was employed for the
identication of impurities. The temperature of the heated capillary
Fig. 1. Chemical structures of griseofulvin and its related impurities. was set at 160 C and the ion spray voltage was set at 4.50 kV. The
capillary voltage was set at 10 V and the tube lens offset voltage at
5 V. Octapole 1 and octapole 2 offset voltages and the interoctapole
validation of such a method was the aim of this research. To be also lens voltages were set at 2.0 V, 7 V and 16 V, respectively. Nitro-
applicable in developing countries, where GF is most administered, gen supplied by Air Liquide (Lige, Belgium) was used as sheath and
it was the intention to use conventional columns and equipment. auxiliary gas at a ow rate of 60 arb. u. and 20 arb. u., respectively.
Helium was used as the damping and collision gas at a pressure of
2. Experimental 0.1 Pa. For MS/MS investigation, the protonated molecule was iso-
lated in the ion trap with an isolation width of 3 u and activated
2.1. Chemicals and reagents at a collision energy level (CEL) of 30%. LCQ Tune software (Ther-
moFinnigan) was used for instrument control, data acquisition and
HPLC gradient grade acetonitrile (ACN) was obtained from processing.
Fisher Scientic (Leicestershire, UK) and methanol (MeOH) from
Acros Organics (Geel, Belgium). Stabilized tetrahydrofuran (THF), 2.4. Robustness study
acetic acid and potassium dihydrogen phosphate (KH2 PO4 ) were
obtained from Merck (Darmstadt, Germany). Phosphoric acid A robustness study was performed by means of an experimental
(H3 PO4 ) was obtained from BDH (Briare, France) and formic acid design and multivariate analysis using Modde 5.0 software (Umet-
(FA) from Biosolve Ltd. (Valkenswaard, The Netherlands). A Milli-Q rics, Ume, Sweden). Experimental factors that might potentially
water purication system from Millipore (Bedford, MA, USA) was cause variability in the method responses were tested. Four factors
used to purify further demineralised water. (column temperature, pH of the mobile phase, amount of ACN
and percent of formic acid) were investigated. A two-level full
factorial design was applied with a number of runs equal to 2k + n,
2.2. Samples and preparation of standard solutions
where k is the number of factors and n is the number of centre
points. Thus, 19 experiments including 3 at the centre point were
The drug substance utilized for method development was an old
performed in triplicate. The lower and higher values for each
GF sample (15 years old) from LUDECO (Brussels, Belgium). This old
factor in the design are given in Table 1. In every experiment, the
sample was used in order to include as much as possible impurities
percent of the 3 potential main impurities of GF (griseofulvic acid,
from fermentation and degradation. For assay, GF drug substance
obtained from SA Aca pharma NV (Waregem, Belgium) was used.
Eight different commercial GF tablet samples were sourced from Table 1
retail pharmacies in Ibadan, Nigeria. For investigation, a 0.5 mg/mL Chromatographic parameter settings applied in the robustness investigation, corre-
sponding to low (), central (0) and high (+) levels.
solution of GF was made in the mobile phase. For the assay of
commercial tablets, a corresponding weight was taken. A 1.0% dilu- Parameter Low value () Central value (0) High value (+)
tion was used as reference for the quantication of the related Temperature ( C) 28 30 32
substances. Samples were ltered using a Chromal Xtra mem- pH 4.3 4.5 4.7
brane lter, 0.2 m obtained from Macherey-Nagel GmbH (Dren, Acetonitrile in B (%, v/v) 64 65 66
Germany). % Formic acid 0.05 0.06 0.07
G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917 11

Table 2
Columns utilized with their characteristics as specied by manufacturers.

Name of column Manufacturer/supplier Dimension (mm mm) Particle size (m) End-capped Base-deactivated F-values

Hypersil BDS C18 ThermoQuest 250 4.6 5 Yes Yes 0.000


XTerra RP18 Waters 250 4.6 5 Yes 2.104
Discovery C18 Supelco 250 4.6 5 Yes 0.404
Brava BDS C18 Alltech 250 4.6 5 Yes Yes 0.152
ACE C18 Achrom 250 4.6 5 Yes Yes 0.431
Polaris C18 -A Varian 250 4.6 5 Yes 0.615
Pursuit C18 Varian 250 4.6 5 Yes 0.640
EverestTM C18 monomeric Grace 250 4.6 5 2.203
Zorbax Extend C18 Agilent 250 4.6 5 Yes 2.137

: not specied.

dechlorogriseofulvin and dehydrogrisofulvin) and the resolution coupling of LC with MS would be interesting. Unfortunately, a phos-
between the two critical peaks (GF and dehydrogrisofulvin) were phate buffer is not compatible with MS, but replacement by 0.1%
considered as response. The statistical relationship between a formic acid and adjustment of the pH to 4.5 could also well separate
response y and the experimental variables xi , xj , . . . is as follows: all components. Thus, using mobile phases A (water0.1% formic
acid pH 4.5, 80:20, v/v) and B (ACNwater0.1% formic acid pH 4.5,
y = 0 + i xi + j xj + ij xi xj + + E (1) 65:15.20, v/v/v) and the gradient described in Section 2.3.1, a good
separation was obtained within 20 min.
where the various -values are the regression coefcients and E
is the overall experimental error. The linear coefcients i and j
describe the quantitative effect of the experimental variables in
3.1.2. Selection of stationary phase
the model while the cross coefcient, ij , measures the interaction
The performance characteristics of 8 other C18 columns apart
effect between the variables i and j.
from Hypersil BDS were investigated using the chromatographic
conditions obtained so far. The performance characteristics of the
3. Results and discussion columns were mainly assessed for their ability to separate GF and
dehydrogriseofulvin (critical peak pair), as the early and late eluting
3.1. Method development and optimization peaks were not signicantly affected. The various columns uti-
lized and their specications from manufacturers are presented in
In the development of a method for the analysis of GF and its Table 2. Retention factor, selectivity, peak asymmetry and reso-
impurities, several parameters were investigated and optimized: lution were the parameters assessed. It was observed that all C18
detection wavelength, effect of organic modiers, effect of pH of columns were not equally effective in separating the critical peaks.
mobile phase, effect of ow rate, effect of concentration of buffer Among the columns examined 3 columns (Hypersil BDS C18 , Dis-
solution, effect of column temperature, effect of injection volume covery C18 and Brava BDS C18 ) gave similar separations of GF and its
and gradient elution programmes. Ten silica based C18 columns impurities. These columns are also indicated to give similar results
were selected for the method development based on the col- according to the column classication system [32]. The best resolu-
umn classication system previously developed in our laboratory tion for the critical peaks was obtained on the Discovery C18 which
[2933]. The classication is based on the determination of four was thereafter used for further optimization of the method. The 6
chromatographic test parameters for each column. A ranking versus remaining columns gave either some peak tailing or fronting and
an arbitrary chosen reference column can be obtained by calculat- thus the overall separation was less satisfactory.
ing F-values. The F-values express the similarity of a column to the
reference column. The smaller the F-value, the more similar is the
column towards the reference column. The various columns uti- 3.1.3. Further optimization of the method
lized and their specications from manufacturers are presented in The developed method was further optimized for detection
Table 2. wavelength, column temperature, ow rate and injection volume.
Comprehensive investigation of the effect of detection wavelength
3.1.1. Selection of mobile phase from 250 nm to 300 nm showed that all peaks were observed to
First, mobile phases were evaluated which have been ear- have most intense signals at 290 nm. Some earlier reports on the
lier reported in literature either for the analysis of GF in dosage determination of GF have been carried out at ambient temperature.
forms, fermentation media or biological samples. A mixture of As this is ambiguous, different temperatures (2530 C) were inves-
MeOHwater (60:40, v/v) or ACNwater (40:60, v/v) in combina- tigated for their effect on the separation of GF and its impurities. It
tion with a Hypersil BDS column could not properly separate GF was observed that the resolution between GF and dehydrogriseo-
and its impurities. When ACN was combined with 50 mM of acetic fulvin was higher (above 3.0) when the column was immersed in
acid (45:55, v/v) [34], the separation improved, but co-elution of a water bath maintained at 30 C compared to 25 C. A somewhat
GF and dehydrogriseofulvin was observed. This observation led higher temperature is also advisable as the method can be more
to the selection of these two peaks as the critical pair to moni- easily applied then in a tropical climate. The ow rate and injection
tor for further optimization. Next, a phosphate buffer was used to volume were found to be optimal at 1.0 mL/min and 10 L, respec-
explore the inuence of different pH values. A pH of 4.5 was found tively. Chromatograms obtained using the optimized conditions on
to give the better, but still unsatisfactory results. Thereafter, gradi- the Discovery column, but also on a Hypersil BDS and Brava BDS are
ent elution was introduced with different combinations of mobile presented in Fig. 2. As no reference substances for the impurities
phase A (water0.2 M phosphate buffer pH 4.5, 80:20, v/v) and B were available, they were characterized by MS (Section 3.2). Next,
(ACNwater0.2 M phosphate buffer pH 4.5, 65:15:20, v/v/v). This the method was validated for its robustness, sensitivity, linearity,
way, selectivity was improved and the critical peak pair was sep- precision and accuracy based on the ICH guidelines (Section 3.3)
arated with a resolution greater than 2.5. For peak identication, [35].
12 G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917

50,0
mAU WVL:290 nm

GF
40,0
Imp 3

Imp 6
Imp 4
30,0 Imp 1 Imp 2

Imp 5 Discovery

20,0

Brava BDS
10,0

Hypersil BDS

-5,0 min

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0

Fig. 2. Chromatograms obtained from the three C18 (250 mm 4.6 mm, 5 m) columns showing overall similar separations of GF and the impurities from a 0.5 mg/mL solution
of griseofulvin drug substance using the nal method. Chromatographic conditions: mobile phase A (water0.1% formic acid pH 4.5, 80:20, v/v) and B (ACNwater0.1%
formic acid pH 4.5, 65:15:20, v/v/v), ow rate 1.0 mL/min, column temperature 30 C, injection volume 10 L and UV detection at 290 nm. Imp: impurity and GF: griseofulvin.

3.2. Characterization of impurities by MS neutral loss of CH3 resulting in [M+H]+ of m/z 336.0. Another less
abundant fragment ion of m/z 291.1 was also formed as shown in
GF is mainly produced by fermentation, thus it is not included Fig. 3. So, impurity 4 is proposed to be dehydrogriseofulvin.
in the ICH Q3 guidelines which set thresholds for the identi-
cation, reporting and qualication of related impurities in active 3.3. Method validation
substances manufactured by chemical synthesis. According to
the European Medicines Agency (EMA) guideline [36] on setting 3.3.1. Robustness study
specications for related impurities in antibiotics produced by Variations in LC operating conditions were made deliberately
fermentation, the reporting threshold is set at 0.10% and the identi- to demonstrate the robustness of the method. The effect of small
cation threshold at 0.15%. So, it was decided to identify the related changes of the chromatographic parameters on the results was
impurities which are 0.15% using MS experiments. Except the MS, investigated by means of an experimental design. The different
this required no adaptations to the developed method as it uses chromatographic parameter settings in the design and corre-
already volatile mobile phases. sponding responses investigated are described in Section 2.4. The
Six impurities were found in an old GF sample injected to the LC percentage of three impurities and two resolutions (Rs) between
system. Impurities 2, 3 and 4 were present at a level > 0.15% while closely eluting peaks were considered as response variables: the
impurities 1, 5 and 6 were only present at levels of 0.040.06%. resolution between GF and impurity 4 (Rs 1) and the resolution
To improve the detectability, fractions containing the respective between impurity 5 & impurity 6 (Rs 2). In the regression coef-
unknown peak were collected from the outlet of the LC system cient plot (Fig. 4), the bars denoted by one variable represent
described under Section 2.3.1 after UV detection. Next, the collected the linear effect of that particular variable while the bars denoted
fractions were concentrated using nitrogen gas to reduce the vol- by variable1*variable2 represent the interaction between the two
ume of the solution, followed by direct infusion into the MS system variables indicated (see Eq. (1)). When the error lines ( = 0.05)
using a built-in syringe pump at a ow rate of 50 L/min. Full scan include zero, the variation of the response caused by changing the
MS and MS/MS analysis were done on each impurity and the main variable is smaller than the experimental error and the effect is
peak. considered to be not signicant.
Impurity 2 with a retention time of 6.5 min showed a protonated It was found that the percentages of the smaller impurities 1, 5
molecule [M+H]+ with m/z 339.2. This ion was further fragmented and 6 remained the same when evaluated in the 19 experiments.
at a CEL of 30% to produce the corresponding product ions listed From the regression coefcient plot of the other impurities (2, 3 and
in Table 3. The proposed structures for the ions of the MS and 4), it can be inferred that there is no signicant effect present from
MS/MS experiments are shown in Fig. 3. These structures were pro- any factor or interaction in the investigated range. On the other
posed based on Ballantine and Fenwicks work on the mass spectra hand, ACN has a signicant, but small positive effect on Rs 1. No
of griseofulvin analogues [37]. From the mass of the protonated other signicant effect on Rs 1 was observed from the variables
molecules and structures of the product ions, this impurity was and their interactions. A similar effect was observed for the pH and
found to be griseofulvic acid. temperature on Rs 2. Formic acid and interaction between pH and
Similarly, impurity 3 with a retention time of 12.4 min showed acetonitrile showed a small negative effect on Rs 2. No other fac-
a [M+H]+ of m/z 319 and four product ions after fragmentation tors or interactions were found to have an important effect on Rs
as indicated in the same gure. Analysis of the precursor ion and 2. In order to better estimate how the variables affect the response
its fragments revealed that this impurity is dechlorogriseofulvin. over the experimental domain, response surface plots (Fig. 5) for Rs
Townley [3] reported that dechlorogriseofulvin is commonly found 1 and Rs 2 were constructed using Modde software. The response
in commercial batches of GF in the range from 0.5 to 3.5%. In this surface plot is a graph of the response function as a function of the
study, 2 drug substances and 8 commercial GF tablets were ana- variables (the main effects observed) while the other parameters
lyzed (see Section 3.4) and it was found that impurity 3 was present are kept constant at their central values. In the ranges examined
in each of the samples in the range from 0.7 to 2.6% and is the major Rs1 is always above 2.92 and the minimum resolution for Rs 2 was
impurity. found to be 1.5. This shows that the peak pairs will always be well
Impurity 4 with a retention time of 17.7 min showed a [M+H]+ separated in the examined range. It can be concluded that small
of m/z 351.5. Fragmentation of this ion showed only an abundant variations in the investigated chromatographic conditions will not
G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917 13

Table 3
Precursor and most abundant product ions of GF, griseofulvic acid, dechlorogriseofulvin and dehydrogriseofulvin produced using product ion scan (Full scan MS and MS2 ).
The structures are illustrated in Fig. 3.

Compound

GF Dechlorogriseofulvin Griseofulvic acid Dehydrogriseofulvin

Protonated molecules (m/z) 353.4 319.0 339.2 351.5

Product ions (m/z) 165.3 165.3 229.0 291.1


215.3 181.2 255.1 336.0
285.1 251.1 297.0
321.0 287.1

The most abundant product ion of each compound is given in bold.

have a detrimental effect on the % of impurities and their resolu- ratio (S/N) of 3 and 10, respectively. An LOD value of 0.06 g/mL,
tions indicating that the developed method is robust. corresponding to 0.01% and an LOQ value of 0.2 g/mL, correspond-
ing to 0.04% of the total GF injected (100% = 500 g/mL) were found.
3.3.2. Quantitative aspects
3.3.2.1. Sensitivity. The limit of detection (LOD) and limit of quan- 3.3.2.2. Linearity. Linearity of the detector response was exam-
tication (LOQ) for GF were determined based on a signal-to-noise ined for assay and for related substances. For the assay method,

Fig. 3. Proposed structures for the ions after fragmentation of the protonated molecule at 30% CEL. The MS conditions are described under Section 2.3.2. Griseofulvic acid,
dechlorogriseofulvin and dehydrogriseofulvin are impurities 2, 3 and 4 in Fig. 2, respectively.
14 G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917

% imp 2

0.010
0.008
Regression coefficients

0.006
0.004
0.002
0.000
-0.002
-0.004
-0.006
-0.008
Temp

pH

ACN

FA

Temp*pH

Temp*ACN

Temp*FA

pH*ACN

pH*FA

ACN*FA
Variables and interactions

% imp 3 Rs 1 (GF - imp 4)

0.06

Regression coefficients
Regression coefficients

0.10
0.04

0.02 0.05

0.00
0.00
-0.02

-0.04 -0.05

-0.06
-0.10
Temp

pH

ACN

FA

Temp*pH

Temp*ACN

Temp*FA

pH*ACN

pH*FA

ACN*FA

Temp

pH

ACN

FA

Temp*pH

Temp*ACN

Temp*FA

pH*ACN

pH*FA

ACN*FA
Variables and interactions Variables and interactions

Rs 2 (imp 5 - imp 6)
% imp 4

0.02 3.0
Regression coefficients
Regression coefficients

0.01
2.0

0.00

1.0
-0.01

0.0
-0.02
Temp

pH

ACN

FA

Temp*pH

Temp*ACN

Temp*FA

pH*ACN

pH*FA

ACN*FA

Temp

pH

ACN

FA

Temp*pH

Temp*ACN

Temp*FA

pH*ACN

pH*FA

ACN*FA
Variables and interactions Variables and interactions

Fig. 4. Regression coefcient plots obtained from the robustness study. Rs 1 (GFimp 4): resolution between griseofulvin and impurity 4, Rs 2 (imp 56): resolution between
impurities 5 and 6 and % imp: percent of impurity, temp: column temperature, ACN: percent of acetonitrile and FA: percent of formic acid.

six concentrations (125 g/mL, 250 g/mL, 375 g/mL, 500 g/mL, 3.3.2.3. Precision. Precision of the developed method was eval-
625 g/mL and 750 g/mL) were prepared from 25% to 150% with uated as intra- and inter-day precision on the peak areas and
respect to the analyte concentration of 500 g/mL. For the related reported as %RSD [38,39]. The intra-day precision was deter-
substances test, griseofulvin concentrations ranging from LOQ to mined by repeatedly analysing six independent solutions prepared
50 g/mL (=10% of the test solution) were examined. To assess according to the method procedure at a concentration of 0.5 mg/mL
the appropriateness of using a linear regression model to t the on the same day. Six of the potential impurities were present at con-
data, residual plots were produced. The points in these plots were centrations 0.04%. Intra-day precision for the main peak GF was
randomly distributed around the horizontal zero axis suggesting found to be 0.02% (n = 6) and not more than 4.0% for the related
that the linear model gives a good t of the data. Results for the impurities. The intermediate precision of the method was veried
regression equation (y), standard error of estimate (Sy,x ), the cor- by comparing results of three consecutive days (Days 13) and the
relation coefcient (r), the 95% condence interval of the intercept third and fourth day (Days 34) where the results of day 4 were
of GF and related substances are shown in Table 4. The 95% con- obtained by a second analyst on a different LC equipment. New
dence interval of the intercept includes zero so that the intercept is preparations, as for intra-day precision, were made on each day for
statistically not signicant. The results indicated that a linear rela- the determination of the intermediate precision. Results obtained
tionship between the detector response and concentrations exists are summarized in Table 5. The % RSD values for the intermediate
in the range examined. precision were not more than 4.6% for the impurities and only 0.1%

Table 4
Regression data for griseofulvin in two concentration ranges, 25150% and LOQ10%.

Range Regression equation

Slope Intercept Interval of the intercept ( = 0.05) Correlation coefcient (r) Sy ,x a

Lower 95% Upper 95%

LOQ10% 2.7925 0.0478 0.12423 0.21993 >0.9999 0.09


25150% 2.5322 5.3298 1.37349 12.03299 0.9996 2.59
a
Sy,x : standard error of estimate.
G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917 15

A. Rs 1 (GF - imp 4)

B. Rs 2 (imp 5- imp 6)

Fig. 5. Response surface plots showing the inuence of temperature and acetonitrile on Rs 1 (A) and pH and temperature on Rs 2 (B). The other parameters are kept constant
at their central values.

for GF. The low RSD values demonstrate the good precision of the 160 g/mL). The determination was carried out using three repli-
method. cates at each concentration level. Accuracy was determined as
percent recovery of the known added amount of the standard to
3.3.2.4. Accuracy. Accuracy of the developed method was deter- the sample. It was found that the method gave a mean recovery of
mined by the standard addition method on the drug product 100.4% (RSD = 0.8%, n = 4) for GF indicating that the sample excipi-
(500 g) to which known amounts of GF have been added at ents do not have effect or interference on the quantication of GF
different concentrations (40 g/mL, 80 g/mL, 120 g/mL and in the drug products.
16 G. Kahsay et al. / Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 917

50,0
mAU WVL:290 nm

Imp 1: unknown
GF
Imp 2: griseofulvic acid
40,0
Imp 3 Imp 3: dechlorogriseofulvin
GF : griseofulvin
30,0 Imp 4: dehydrogriseofulvin
Imp 5: unknown
Imp 6: unknown
20,0

10,0
Imp 6
Imp 2 Imp 4
Imp 1 Imp 5

-5,0 min

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0

Fig. 6. Typical chromatogram obtained for a 0.5 mg/mL solution of griseofulvin tablets using the nal method. Chromatographic conditions: Discovery C18 (250 mm 4.6 mm,
5 m) column, mobile phase A (water0.1% formic acid pH 4.5, 80:20, v/v) and B (ACNwater0.1% formic acid pH 4.5, 65:15:20, v/v/v), ow rate 1.0 mL/min, column
temperature 30 C, injection volume 10 L and UV detection at 290 nm.

Table 5 for sensitivity, linearity, precision and accuracy in the investi-


Results of the intermediate precision determination for GF and its impurities (Imp)
gated range based on the ICH guidelines. A robustness study was
expressed as % RSD.
performed by means of an experimental design and multivariate
Number of days Imp 1 Imp 2 Imp 3 GF Imp 4 Imp 5 Imp 6 analysis. Results showed that the method was sensitive, linear,
Day 1 (n = 6) 3.2 1.5 0.6 0.02 1.6 4.0 3.9 precise, accurate and robust in the studied ranges. Three impuri-
Day 2 (n = 6) 4.0 1.4 0.4 0.02 0.8 3.0 3.0 ties present above the identication threshold [36] were identied
Day 3 (n = 6) 2.3 1.7 0.4 0.02 1.4 3.1 2.5 using MS experiments. The developed LC method has been applied
Days 13 (n = 18) 3.9 1.6 0.8 0.03 1.5 3.3 4.3
for the assay and impurity determination of GF drug substances and
Day 4 (n = 6) 2.3 1.1 0.4 0.02 2.0 2.3 3.2
Days 34 (n = 12) 2.3 2.1 2.6 0.1 1.8 2.6 4.6 tablets commercially available on the market. Moreover, the new LC
method is suitable to be coupled with MS for further identication
and characterization of GF impurities.
Table 6
Results of impurity determination for griseofulvin tablets (samples AH) and drug
substance samples (IJ). Acknowledgements
Sample Impurities (expressed as GF, %m/m)
The authors would like to thank the Interfaculty Council for
Imp 1 Imp 2 Imp 3 Imp 4 Imp 5 Imp 6 Development Co-operation (IRO), Katholieke Universiteit Leuven,
sample A <LOQ 0.33 2.06 0.39 <LOQ <LOQ Belgium, for support. A.O. Adegoke was a visiting fellow in the
sample B ND 0.23 1.76 0.39 ND ND framework of a Coimbra scholarship.
sample C 0.05 0.44 2.34 0.50 ND ND
sample D <LOQ 0.40 1.58 0.48 ND ND
sample E 0.04 0.58 1.72 0.29 ND ND References
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