27-09-2012, 05:41 PM
ICH guidance in practice: Validated stability-indicating HPLC method for simultaneous determination of ampicillin and cloxacillin in combination drug products
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Abstract
Ampicillin and cloxacillin were degraded together under different stress test conditions prescribed by International Conference on Harmonization.
The samples so generated were used to develop a stability-indicating high performance liquid chromatographic (HPLC) method for
the two drugs. The drugs were well separated from degradation products using a reversed-phase (C-18) column and a mobile phase comprising
of acetonitrile:phosphate buffer (pH 5.0), which was delivered initially in the ratio of 15:85 (v/v) for 1 min, then changed to 30:70 (v/v)
for next 14 min, and finally equilibrated back to 15:85 (v/v) from 15 to 20 min. Other HPLC parameters were: flow rate, 1 ml/min; detection
wavelength, 225 nm; and injection volume, 5 l. The method was validated for linearity, precision, accuracy, specificity and selectivity. It was
also compared with the assay procedures given in British Pharmacopoeia for individual drugs. Similar results were obtained, indicating that
the proposed single method allowed selective analysis of both ampicillin and cloxacillin, in the presence of their degradation products formed
under a variety of stress conditions. The developed procedure was also applicable to the determination of instability of the drugs in commercial
products.
Introduction
In recent times, there is an increased tendency towards the
development of stability-indicating assays [1–3], using the
approach of stress testing as enshrined in the International
Conference on Harmonization (ICH) guideline Q1AR(2) [4].
Even this approach is being extended to drug combinations
[5,6], to allow accurate and precise quantitation of multiple
drugs, their degradation products, and interaction products,
if any.
The combination containing ampicillin and cloxacillin shows
synergistic effect [7] and is extensively used for the treatment
of infections in premature infants and newborns [8]. It is also
employed for therapy of resistant infections, and for veterinary
purposes [9]. There is no stability-indicating assay method
reported yet for this combination, developed using the ICH
approach of stress testing. Otherwise, there are several HPLC
procedures known for the analysis of ampicillin [10–12] and
cloxacillin [13–15] individually, and some methods even exist
for simultaneous analysis of the two drugs, either in a veterinary
combination [16] or biological fluids [17,18]. Recently, we also
developed a derivative spectrophotometric method targeted to
selective analysis of ampicillin and cloxacillin in drug combination
[19]. The procedure gave acceptable results with fresh
products, but gave overestimation during analysis of stability
samples and aged products.
Equipment
The HPLC system consisted of an on-line degasser
(DGU-14A), low-pressure gradient flow control valve (FCV-
10ALVP), solvent delivery module (LC-10ATVP), auto injector
(SIL-10ADVP), column oven (CTO-10ASVP), UV–vis dualwavelength
detector (SPD-10AVP), photo-diode array (PDA)
detector (SPD-M10AVP), system controller (SCL-10AVP) and
CLASS-VP software, ver. 6.13 (all from Shimadzu, Kyoto,
Japan). The separations were achieved on a inertsil ODS2
column (250mm×4.6 mm, 5m) from Flexit Jour, Pune,
India and grace vydac C-18 (250mm×4.6 mm, 5m) column
from Hesperia, CA, USA. The latter was used for intermediate
precision studies. A precision water bath equipped with MV
controller (Julabo, Seelbach, Germany) was used to carry out
selected reactions in solution. Stability studies were carried out
in humidity (KBF720, Binder, Germany) and photostability
(KBWF240,WTCBinder, Germany) chambers both set at 40 ◦C
±1 ◦C/75% RH±3% RH.
Validation of the method
The method was validated for linearity, precision (inter-day,
intra-day and intermediate precision), accuracy, specificity and
selectivity. Standard plots were constructed for both ampicillin
and cloxacillin in the range of 50–1000 g/ml. The experiment
was repeated thrice on the same day and additionally on two consecutive
days to determine intra- and inter-day precision, respectively.
The intermediate precision of the method was determined
by repeating the experiment on two different columns. Accuracy
was determined by fortifying the mixture of degraded solutions
with three known concentrations of the drugs. Further, specificity
of the method was assessed by study of the resolution
factor of the drug peaks from nearest resolving peaks. The selectivity
was determined by checking peak purity of all the peaks,
including those of degradation products, using a PDA detector
Conclusion
This study presents a simple and validated stability-indicating
HPLC method for simultaneous estimation of ampicillin and
cloxacillin in the presence of degradation products. The method
yielded results similar to those determined by application of
compendial procedures on individual drugs. The method could
be applied with success even to the analysis of marketed products,
as no interference was observed due to excipients or other
components present.