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Hidroclorotiazidă HPLC - Grupa 23
Hidroclorotiazidă HPLC - Grupa 23
of a Stability-Indicating LC Method
to Quantify Hydrochlorothiazide
in Oral Suspension for Pediatric Use
2008, 67, 647–652
Monika Piazzon Tagliari&, Hellen Karine Stulzer, Fabio Seigi Murakami, Gislaine Kuminek,
Bruno Valente, Paulo Renato Oliveira, Marcos Antonio Segatto Silva
Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina (UFSC),
Campus Universitário Trindade, Bloco K, sala 207, Florianópolis, SC 88040-900, Brazil; E-Mail: monikatag@yahoo.com.br
Full Short Communication Chromatographia 2008, 67, April (No. 7/8) 647
DOI: 10.1365/s10337-008-0546-1
0009-5893/08/04 2008 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
Although stability data for extempo- with a 20 lL loop. The detector was set plate during 60 s to assure homogeneity
raneous suspensions of some antihyper- at 254 nm and peak areas were inte- at the time to take the aliquot. An ali-
tensive agents like lisinopril, captopril grated automatically by computer using quot of 0.3 mL were pipetted from the
and quinopril have been published, a Shimadzu Class VP V 6.14 software suspension and quantitatively trans-
[2, 4, 15] such suspensions have inherent program. The experiments were carried ferred in to a 25 mL volumetric flask
problems, including limited stability, out on a reversed-phase Phenomenex containing 2 mL of methanol, and stir-
questionable uniformity, and unknown (Torrance, USA) Luna C18 column red in an ultrasonic bath for 10 min. The
bioavailability [16]. There is no com- (250 mm · 4.6 mm I.D., with a particle volume was completed with the mobile
mercially available formulation of HCTZ size of 5 lm and pore size of 100 Å), phase (30 lg mL 1), and the resulted
oral suspension, because of its instability maintained at 40 ± 1 C. A security solution was filtered through a 0.45 mm
in aqueous solution and therefore the guard holder (4.0 mm · 3.0 mm I.D.) nylon membrane.
development of a liquid formulation is a was used to protect the analytical col-
significant challenge [17]. umn. The mobile phase consisted of
A new pharmaceutical formulation 0.1 M sodium phosphate buffer pH 3.0 Preparation of Standard
of HCTZ suspension has been developed and acetonitrile (70:30 v/v), and was Solutions
and to assure its quality for further eluted isocratically at a flow rate of
studies in vivo (protocol already 1.3 mL min 1. A stock standard solution of 1,000 lg
approved CEPSH-UFSC: 259/06), a mL 1 was prepared by dissolving 25 mg
stability-indicating analytical method of the HCTZ reference standard in 2 mL
should be developed and validated. At Chemicals of acetonitrile and 10 mL of mobile
the moment, there is no published phase in a 25 mL volumetric flask, and
method validated for the quantitative The HCTZ reference standard stirred in an ultrasonic bath for 10 min.
analysis of HCTZ in suspension, and (99H1207) with stated purity of 100.1% The volume was completed with the
even with methods published for its and chlorothiazide (CTZ, 52K1441) same mobile phase. A stock solution of
determinations in solid pharmaceutical were obtained from Sigma–Aldrich CTZ at 1,000 lg mL 1 was also pre-
formulations, the applicability of the (Steinheim, Germany). The HCTZ raw pared in the mobile phase.
methodologies to pharmaceutical sus- material was donated by LAFESC
pensions must be evaluated demonstrat- (Laboratório Industrial Farmacêutico de
ing the validation parameters, including Santa Catarina, Florianópolis, Brazil). Method Validation
the specificity of the methods towards Ultra-pure water was provided by a
potential degradation products and for- Milli-Q purification system (Millipore, Analytical method development and
mulation excipients. Liquid chromatog- Bedford, USA). Methanol and acetoni- validation play a major role in the dis-
raphy with UV detection has been used trile of LC grade were purchased from covery, development, and manufacture
for quality control of most of the phar- Vetec (Rio de Janeiro, Brazil). All of pharmaceuticals [18]. The Interna-
maceuticals due to its simplicity, high chemicals used were of pharmaceutical tional Conference on Harmonization
resolution and significant precision and or special analytical grade. (ICH) [19] requires the stress testing to
accuracy. The aim of the present work be carried out to elucidate the inherent
was to develop and validate a stability- stability characteristics of the active
indicating LC method for the determi- Production of HCTZ substance. An ideal stability-indicating
nation of HCTZ in a pharmaceutical Suspension method quantifies the drug per se and
oral suspension. also resolves its degradation products
HCTZ raw material was crushed in a [20].
Mortar with a pestle to get smaller par- The method was validated to quan-
Experimental ticle sizes. As part of this process, the tify the HTCZ in a pediatric suspension
particles were individually wetted with by the determination of the following
Instruments and Analytical glycerin, and a polymeric solution of parameters according to the ICH [19]:
Conditions carboxymethylcellulose sodium (0.6%) specificity, linearity, accuracy, precision,
containing sodium benzoate (0.1%) as a robustness, quantification and detection
The LC analysis was performed on a preservative was added. The final for- limits.
Shimadzu LC-10A system (Kyoto, mulation was kept in stability pH 3.2.
Japan) equipped with an LC-10AD
System Suitability
pump, SPD-10AVVP UV detector,
SPD-M10AVP photodiode array (PDA) Preparation of Sample The system suitability was carried out to
detector, SCL-10AVP system controller, Solutions evaluate the resolution and reproduci-
DGU-14A degasser, CTO-10ASVP col- bility of the system for the analysis to
umn oven, and the sample injection was Before the measurements, each suspen- be performed, using six replicate ana-
performed via a Rheodyne 7125 valve sion vial was stirred on a magnetic stir lyses of the drug at a concentration of
648 Chromatographia 2008, 67, April (No. 7/8) Full Short Communication
30 lg mL 1, evaluating the following
parameters: peak area, retention time,
asymmetry and theoretical plates.
Specificity
Full Short Communication Chromatographia 2008, 67, April (No. 7/8) 649
HCTZ retention time was about 3.5 min,
and a typical chromatogram obtained by
the proposed method is shown in Fig. 1a.
The system suitability results showed
that the parameters are within the suit-
able range. The mean asymmetry found
was 1.17 with RSD (%) of 0.38. The
mean theoretical plates, retention time
and peak area ± RSD (%) found were
9,749 ± 1.11, 3.559 ± 0.08, and
504,966 ± 0.57, respectively.
Method Validation
Specificity
650 Chromatographia 2008, 67, April (No. 7/8) Full Short Communication
thermal stress conditions there was no Table 1. Results from determination of the intra- and inter-day precision, and the accuracy of
change in the area and no additional the method
peak was detected after 72 h. % Recovered RSD (%)
The studies with the PDA detector
showed that the HCTZ peaks were free Intra-day precision
from any coeluting peak, with values of Day 1 (n = 6) 99.6 0.44
Day 2 (n = 6) 99.3 0.51
a peak purity index higher than 0.9999, Day 3 (n = 6) 98.4 0.80
thus demonstrating that the proposed Inter-day precision (n = 18) 99.4 0.98
method is specific. Accuracy (n = 3)
Fortified solution Mean concentration Recovery (%)
(lg mL 1) found (lg mL 1) (% RSD)
Linearity, and Limits of Detection (LOD) 15 14.93 98.6 (0.3)
and Quantitation (LOQ) 30 30.34 101.7 (0.6)
45 45.21 100.6 (0.5)
The linearity of detector response was
assessed for various solution standards RSD Relative standard deviation
over the range of 10–50 lg mL 1. The
value of the determination coefficient
Table 2. Chromatographic conditions and range investigated during robustness testing
calculated (r2 = 0.9998, y = 15895.67
x 69.87; where, x is concentration and Variable Range investigated Hydrochlorothiazidea %
y is the peak absolute area) indicated the
linearity of the calibration curve for the Flow rate (mL min 1) 1.2 101.80
1.3 100.12
method. The validity of the assay was 1.4 99.22
verified by analysis of variance, ANOVA Column temperature (C) 35 100.70
(Fcalculated = 8568.5 > Fcritical = 0.0001; 40 100.12
45 100.69
P = 5%).
Percent acetonitrile 25 99.89
The LOQ and LOD calculated were 30 100.12
1.23 and 0.40 lg mL 1, respectively, 35 100.90
which were confirmed experimentally, Mobile phase pH 2.7 100.80
3.0 100.12
indicating adequate sensitivity of the 3.3 100.30
method. Solution stability (48 h) – 99.80
a
Mean of three replicates
Accuracy and Precision
The accuracy was assessed from three determination of the drug in the phar- towards specificity, linearity, accuracy,
replicate determinations of three differ- maceutical formulations. No significant precision, robustness, and was proved to
ent fortified solutions. No significant changes were observed in the content of be capable of separating HCTZ and its
differences were observed between HCTZ during solution stability experi- major degradation product, CTZ,
amounts of HCTZ added and the ment, which confirms that the sample demonstrating its suitability for use as a
amounts found (P < 0.05). The ob- solutions were stable for at least 48 h. stability indicating method during sta-
tained values were within the range of bility studies. The short analytical run
98.0–99.0% (Table 2), satisfying the time of less than 4.0 min leads to a cost-
acceptance criteria for the study. effective and rapid chromatographic
Analysis of HCTZ
The intra-day and inter-day precision procedure. Furthermore, the developed
RSD (%) are given in Table 1. The
in Pharmaceutical Suspension
method showed no interference of the
amounts of HCTZ found on the three formulation excipients and was success-
The results obtained from the validation
different days were equivalent fully applied for the quality control of
studies proved that the method is suitable
(P < 0.05), and the relative standard HCTZ in pharmaceutical suspensions
to quantify HCTZ in the oral suspension.
deviation values were within the accep- for pediatric use.
The quantitative assay of six batches of
tance criteria of 2%.
HCTZ in suspensions for pediatric use
was within 100.91–102.10%.
Robustness References
The results and the experimental range 1. Michele TM, Knorr B, Vadas EB, Reiss
of the variables evaluated in the robust- Conclusion TF (2002) J Asthma 39:391–403
2. Nahata MC Pediatric (1999) Ann Phar-
ness assessment are given in Table 2. The macother 33:247–249
analysis performed resulted in changes in The proposed high-performance liquid 3. Kayumba PC, Huyghebaert N, Cordella
the retention time without effect on the chromatographic method was validated C, Ntawukuliryayo JD, Vervaet C (2007)
Full Short Communication Chromatographia 2008, 67, April (No. 7/8) 651
Eur J Pharmaceut Biopharmaceut 66:460– 10. Carlucci G, Palumbo G, Mazzeo P, 17. Trissel L (2005) In: Trissel’s stability of
465 Quaglia MG (2000) J Pharm Biomed Anal compounded formulations. American
4. Freed AL, Silbering SB, Kolodsick KJ, 23:185–189 Pharmaceutical Association, Washington
Rossi DT, Mahjour M, Kingsmill CA 11. Erk N (1999) J Pharm Biomed Anal 18. Shabir GA, Lough WJ, Arain SA,
(2005) Int J Pharm 304:135–144 20:155–167 Bradshaw TK (2007) J Liq Chromatogr
5. Litwin M, Sladowska J, Antoniewicz J, 12. Dinç E, Özdemir A (2005) Il Farmaco Related Technol 30:311–333
Niemirska A, Wierzbicka A, Daszkowska 60:591–597 19. ICH (2005) Harmonised tripartite guide-
J, Wawer ZT, Janas R, Grenda R (2007) 13. El-Gindy A, Ashour A, Abdel-Fattah L, line: validation of analytical procedures:
Am J Hypertens 20:875–882 Shabana MM (2001) J Pharm Biomed text and methodology Q2 (R1)
6. Moffat A, Osselton M, Widdop B (2004) Anal 25:923–931 20. Vadera N, Subramanian G, Musmade P
In: Clarke’s Analysis of Drugs and 14. Quaglia MG, Donati E, Carlucci G, (2007) J Pharm Biomed Anal 43:722–726
Poisons, Pharmaceutical Press, London Mazzeo, Fanali S (2002) J Pharm Biomed 21. Lusina M, Cindric T, Tomaic J, Peko M,
7. Carter BL, Ernst ME, Cohen JD (2004) Anal 29:981–987 Pozaic L, Musulin N (2005) Int J Pharm
Hypertension 43:4–9 15. Thompson KC, Zhao Z, Mazakas JM, 291:127–137
8. European Pharmacopoeia (2001) 3rd edn, Beasley CA, Reed RA, Moser CL (2003) 22. Hertzog DL, McCafferty JF, Fang X,
p 2966 Am J Health Syst Pharm 60:69–74 Tyrrell RJ, Reed RA (2002) J Pharm
9. The United States Pharmacopoeia (2007) 16. Flynn JT, Daniels SR (2006) J Pediatr Biomed Anal 30:747–760
30 Revision. United States Pharmacopeial 149:746–54
Convention, Rockeville, p 2288
652 Chromatographia 2008, 67, April (No. 7/8) Full Short Communication