Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011 713
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Development and Validation of an HPLC Method for Determination of Ziprasidone and Its Impurities in Pharmaceutical Dosage Forms Marija Pavlovic and Marija Malesevic Medicines and Medical Devices Agency of Serbia, Belgrade, Serbia Katarina Nikolic and Danica Agbaba1 University of Belgrade, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Vojvode Stepe 450, PO Box 146, 11000 Belgrade, Serbia Ziprasidone is known as a novel “atypical” or “second-generation” antipsychotic drug. A sensitive and reproducible method was developed and validated for determination of ziprasidone and its major impurities, which are significantly different in polarity. The separation is performed on a Waters Spherisorb® octadecylsilyl 1 column (5.0 μm particle size, 250 × 4.6 mm id) using a gradient with mobile phase A [buffer–acetonitrile (80 + 20, v/v)] and mobile phase B [buffer–acetonitrile (10 + 90, v/v)] at a working temperature of 25°C. The buffer was 0.05 M KH2PO4 solution with an addition of 10 mL triethylamine/L solution, adjusted to pH 2.5 with orthophosphoric acid. The flow rate was 1.5 mL/min, and the eluate was monitored at 250 nm using a diode array detector. Optimization of the experimental conditions was performed using partial least squares regression, for which four factors were selected for optimization: buffer concentration, buffer pH, triethylamine concentration, and temperature. The proposed validated method is convenient and reliable for the assay and purity control in both raw materials and dosage forms.
Z
iprasidone is a novel “atypical” or “secondgeneration” antipsychotic drug. Orally administered, it is used for the treatment of schizophrenia and the acute manic or mixed episodes associated with bipolar disorder. The short-acting intramuscular ziprasidone mesylate formulation was the first novel antipsychotic drug in the parenteral form for the treatment of acute agitation in patients with schizophrenia (1). This atypical antipsychotic drug has a unique profile, as it acts primarily through serotonergic and dopaminergic receptor antagonism, and as Guest edited as a special report on “New Methods for Drug Analysis in Biological Samples and Other Matrixes” by Danica Agbaba and Andjelija Malenovic. 1 Corresponding author’s e-mail:
[email protected]
an inhibitor of the norepinephrine reuptake (2). It has higher affinity to the 5-HT2A receptor than to the D2 receptor (3). Several methods have been developed enabling pharmacokinetic studies and an assessment of the bioequivalence of ziprasidone in pharmaceuticals. Concentration of ziprasidone (both in human and animal samples) was measured by HPLC with UV (4, 5) and fluorescence (6) detection, and by HPLC coupled with MS (7–12). The metabolism of ziprasidone was investigated in vitro, and the primary oxidative metabolites were identified and quantified by means of HPLC with radioactivity monitoring (13). Only a few studies are reported in the literature that are devoted to the investigation and quantification of ziprasidone in pharmaceuticals and raw materials. The techniques used for this purpose include HPLC-UV (14, 15), capillary zone electrophoresis (16), and HPTLC (17). One recent paper discussed the possible mechanism of ziprasidone oxidation, which was investigated electrochemically (18). Ziprasidone (5-[2-[4-(1,2-benzisothiazol-3-yl)-1piperazinyl]ethyl]-6-chloro-1,3-dihydro-2H-indol2-one) is synthesized by coupling 3-(1-piperazinyl) -1,2-benzisothiazole with 6-chloro-5-(2-chloroethyl)-1,3dihydro-2H-indol-2-one, which are both recognized as impurities related to the synthesis precursors (Impurity I and IV, respectively; Figure 1). Ziprasidone and its one synthetic precursor (Impurity I) are photosensitive in solution due to isomerization of the benzisothiazole moiety to the corresponding benzthiazole. The mechanism of photoisomerization involves the azirine intermediate, and the respective structures were confirmed by means of LC/MS/MS and GC/MS (19). Depending on the reaction conditions applied in the course of the synthesis, appearance of side reactions and consecutive by-product impurities, such as 5-[2-[4-(1,2-benzisothiazol-3-yl)-1-piperazinyl]ethyl]6-chloro-1,3-dihydro-2H-indol-2,3-dione (Impurity II); 5,5΄-bis[2-[4-(1,2-benzisothiazol-3-yl)-1-piperazinyl] ethyl]-6,6΄-dichloro-1,1΄,3,3΄-tetrahydro-3-hydroxy[3,3΄-bi-2H-indole]-2,2΄-dione (Impurity III); and 3-(1,2-benzisothiazol-3-yl)-5-[2-[4-(1,2-benzisothiazol-
714 Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011
for the determination of the five recognized ziprasidone impurities. We proved that the analytical procedure was convenient for the assay and purity control of ziprasidone, both in the bulk drug form and in pharmaceutical preparations. Experimental Chemicals
Figure 1. Chemical structures of ziprasidone and its main impurities.
3-yl)-1-piperazinyl]ethyl]-6-chloro-1,3-dihydro-2Hindol-2-one (Impurity V) could be expected. Reactivity at the α position of the benzoxindol moiety present in the molecule of ziprasidone can be responsible for the instability of ziprasidone, and this may be the reason Impurities II, III, and V differ structurally at this position. Chemical structures of possible ziprasidones impurities are shown in Figure 1. Ziprasidone undergoes considerable degradation in alkaline media and mild degradation under thermal stress conditions. Separation of thermal and baseinduced degradation products was obtained earlier by use of HPLC-UV (20), without structure elucidation of the degradation products. TLC and HPLC methods were then developed for determination of ziprasidone in the presence of the impurities, but without a possibility to provide purity characteristics of the samples studied (21). To the best of our knowledge, none of the methods so far reported has resulted in determination of the drug itself in the presence of its Impurities I–V. Therefore, the aim of this study was to develop an HPLC method
Ziprasidone mesylate trihydrate and Impurities I–V were kindly supplied by Pfizer Inc. (Groton, CT). Zeldox (ziprasidone hydrochloride capsules, 40 mg, and ziprasidone mesylate powder for the solution for injection, 20 mg/mL) were obtained from Pfizer (Illertissen, Germany, and Pocé-sur Cisse, France, respectively). All reagents used in the experiment were analytical purity grade. Water of HPLC purity grade (purified by the Simplicity 185 system; Millipore, Billerica, MA), potassium dihydrogen phosphate (J.T. Baker B.V., Deventer, Holland), and orthophosphoric acid (85%), triethylamine (TEA), and acetonitrile of HPLC purity grade (Merck, Darmstadt, Germany) were used for preparation of the mobile phase. Chromatographic Conditions The Hewlett Packard 1100 Series chromatographic system (Agilent Technologies, Waldbronn, Germany) consisted of a binary pump, diode array detector (DAD), column, thermostated autosampler, degasser, and ChemStation integrator. Separations were carried out using a Spherisorb octadecyl silyl (ODS) 1 chromatographic column (250 × 4.6 mm id, 5.0 μm particle size; Waters, Dublin, Ireland) at 25°C. The injection volume was 40 μL. Solutions were filtered through 0.45 μm Millipore membrane filters (Cork, Ireland). The applied buffer was a 0.05 M solution of potassium dihydrogen phosphate with addition of 1% TEA, pH value adjusted to 2.5 with orthophosphoric acid. Mobile phase A was buffer–acetonitrile (80 + 20, v/v), and mobile phase B was buffer–acetonitrile (10 + 90,
Table 1. Chromatographic parameters of the HPLC assay of ziprasidone and its five impurities Number of theoretical plates (N)
Retention time, min
Compound
Retention factor (k)
Peak asymmetry (As)
Selectivity (α)
Resolution factor (Rs)
—
—
Impurity I
3.7
39575
1.1
0.81
Impurity II
7.3
16528
3.2
1.28
2.22
12.1
Ziprasidone
7.9
50258
3.5
—
1.09
1.55
Impurity III
10.3
72764
4.8
0.81
1.33
8.01
Impurity IV
12.6
57182
6.2
1.08
1.25
6.56
Impurity V
16.1
577085
8.2
0.85
1.29
11.79
Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011 715
concentrated HCl (20 + 5 + 0.01, v/v/v)] in order to obtain a 2.0 mg/mL solution of ziprasidone and 500 mg/mL solutions of Impurities I–V. The solution of Impurity III was stored for 1 day at 4°C, and solutions of ziprasidone and the Impurities I, II, IV, and V were stored for 7 days at 4°C. Because of the photosensitivity of ziprasidone and in order to prevent photodegradation, all solutions were protected from daylight and stored in amber-colored bottles. Solutions for Optimization of the Method Figure 2. Chromatogram of pure ziprasidone sample (1 mg/mL) spiked with 0.3% Impurity II and 0.2% Impurities I, III, IV, and V.
Stock solutions were diluted with the solvent to obtain concentrations of ziprasidone equal to 1 mg/mL; Impurities I and III–V, 2 mg/mL; and Impurity II, 3 mg/mL.
v/v). Prior to use, mobile phases were filtered through a 0.45 mm Millipore membrane filter and degassed in an ultrasonic bath. In the analytical procedure, elution was performed using a combination of the gradient and isocratic elution according to the following scheme: t (min)/%B; 0 min/10%, 6 min/25%, 10 min/25%, 20 min/100%, 20.1 min/10%, and 30 min/10%, with a postanalysis run time of 10 min. The mobile phase flow rate was 1.5 mL/min. Chromatograms were recorded by means of a DAD set at 250 nm. In order to evaluate the suitability of the proposed system, the retention factor (k), peak asymmetry (As), selectivity (α), resolution (Rs), and number of theoretical plates (N) were calculated by use of the Agilent ChemStation software.
Standard Solutions for Testing of Linearity For determination of the calibration curves, a series of seven solutions of ziprasidone were prepared from the stock solution in the concentration range from 70 to 130 mg/mL. Calibration solutions of Impurities I–V were obtained by diluting the respective stock solutions with the solvent to obtain the concentrations within the ranges of 0.06–6.0, 0.06–6.0, 0.08–6.0, 0.07–6.0, and 0.04–6.0 mg/mL, respectively. Sample Solutions (a) Capsule solution.—The content of each of 20 capsules was measured and mixed. A quantity of the mixed content equivalent to 20 mg pure ziprasidone was accurately weighed and transferred into a 20 mL amber-colored volumetric flask. A 10 mL aliquot solvent
Stock Solutions Stock solutions were prepared by dissolving the standard substances in the solvent [methanol–water–
Table 2. Screening results for optimization of the RP-HPLC method for determination of ziprasidone and its impurities Experiment No. 1
Temperature, ºC
pH
KH2PO4, mM
20
2.5
25
Rs-Imp2-Zipra
TEA, %
1
1.32
Rs-Zipr-Imp3b 7.92
2
25
2.5
25
1
1.57
8.16
3
30
2.5
25
1
1.78
8.61
4
25
2.3
25
1
1.54
7.98
5
25
2.5
25
1
1.55
8.01
6
25
2.7
25
1
1.55
8.05
7
25
2.5
25
1
1.55
8.05
8
25
2.5
50
1
1.53
8.3
9
25
2.5
75
1
1.57
8.16
10
25
2.5
25
0.5
1.3
8.76
11
25
2.5
25
1
1.55
8.01
12
25
2.5
25
1.5
1.63
7.49
a
Resolution between ziprasidone and Impurity II.
b
Resolution between ziprasidone and Impurity III.
716 Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011
the leave-n-out (LnO) cross-validation method. The 2 predictive power of the model was determined by Q (the 2 cross-validated R version) and by the root mean square error of prediction. Results and Discussion
Figure 3. Variable importance in the projection (VIP) plots for the response variables, resolution between ziprasidone and its impurities. (a)“Rs Imp2-Zipr” —resolution between ziprasidone and Impurity II. (b)“Rs Zipr-Imp3” —resolution between ziprasidone and Impurity III.
was added to the flask; the obtained solution was mixed in an ultrasonic bath for 15 min and diluted to volume with the solvent. Then the solution was centrifuged at 4000 rpm for 10 min, and the clear supernatant (Solution S, 1 mg/mL) was used for the assay of the impurities. (b) Powder for the injection solution.—The content of one vial with 20 mg pure ziprasidone was completely dissolved in the solvent and quantitatively transferred to a 20 mL amber-colored volumetric flask. Then it was diluted to 20 mL with the solvent to obtain Solution S (1 mg/mL). The procedure was the same as that used for processing of capsules. For the assay of ziprasidone, 1 mL Solution S was transferred to a 10 mL volumetric flask and diluted to volume with the solvent (100 µg/mL). Software and Computations Partial least square regression (PLSR), i.e., the recently developed generalization of multiple linear regression (22), was used for calculation of variable importance in the projection (VIP) and for development of the quantitative structure-activity relationship (QSAR) model. For the regression analysis, the Soft Independent Modeling of Class Analogy (SIMCA) P+ 12.0 program was used (23). The quality of the regression fits was estimated using such parameters as the squared correlation coefficient (R2), the root mean square error of estimation, and Q2(Y) (24). Validation of the PLSR models was performed using
Ziprasidone is not an official active pharmaceutical ingredient in the European Pharmacopoeia or the U.S. Pharmacopeia. Moreover, there are not many publications available on simultaneous quantification of ziprasidone and its Impurities I to V in bulk powder and dosage forms. Therefore, the purpose of this investigation was to develop and validate a selective RP-HPLC method for the analysis of ziprasidone and its five impurities that differ in polarity. Ziprasidone, a highly hydrophobic drug, has an ability to pass through the blood brain barrier, an important prerequisite for the therapeutic effects. Due to the presence of the piperazine moiety, ziprasidone has basic properties. Figure 1 shows that Compounds I and IV originate from the synthesis as the precursor impurities. All the impurities, except for Impurity IV, are basic; Impurity IV does not have basic properties, as it lacks the piperazine part, and its reactivity is due to the presence of the benzyl halide functionality, which could be responsible for mutagenicity (25). Impurities II, III, and V are the degradation products of ziprasidone. Ziprasidone and Impurity II are the most structurally similar, the latter being the oxidation product of ziprasidone with an additional keto group in the α position of the benzoxindol moiety. Due to the earlier mentioned reactivity of this position, one can assume that the occurrence of Impurity III might result from coupling of Impurity II with ziprasidone. Impurity V elutes as the last peak due to the additional benzisothiazol moiety at the α position of ziprasidone, which significantly increases its lipophilicity. In order to develop and validate a sensitive and selective analytical method, different options were evaluated to optimize the chromatographic conditions. For this purpose, the combined effect of the elution mode, pH value, and composition of the mobile phase on the RPLC behavior of ziprasidone and its five impurities was investigated. In previously published papers, the chromatographic behavior of ziprasidone was investigated on C18 chemically bonded stationary phases using the isocratic (14, 15, 21) and gradient (5, 20) elution modes. Recently, a significant improvement has been achieved in the quality of HPLC chemically bonded silica particles, which resulted in an increased number of the available RP columns with different functionalities and selectivities. Due to the aforementioned difference in the polarity of ziprasidone and Impurities I–V, our study was carried out using several different stationary phases with a wide range of hydrophobicity, i.e., the less hydrophobic cyanopropyl (CN) and amino (NH2) chemically
Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011 717
bonded stationary phases and the more hydrophobic phenyl, octyl (C8) and C18 phases. Due to structural similarity of ziprasidone and Impurity II development of a chromatographic method able to separate these two compounds caused analytical problems; resolution of this particular pair proved decisive for the choice of the least polar C18 stationary phase. Satisfactory resolution and elution time (the last Impurity V peak eluted in 13.5 min) was achieved on a densely bonded and double endcapped Zorbax Eclipse XDB C18 column (250 × 4.6 mm id, 5 µm particle size; Agilent). However, with this column, the As of Impurity II was higher than 2, with significant peak tailing. In order to improve peak symmetry and resolution, the same gradient conditions were applied with a better-performing Spherisorb ODS 1 column (250 × 4.6 mm, 5 µm particle size; Waters). The main difference between these columns was that the Spherisorb ODS 1 is an unendcapped C18 column with less-hydrophobic properties (26, 27), which results in a decrease of the peak tailing and subsequent improvement of the peak symmetry. Due to its better chromatographic performance, the Waters Spherisorb ODS 1 column (characterized by spherical particle shape and greater specific surface area than the Zorbax Eclipse XDB C18 packing) was selected for the development and validation of the new method of determination ziprasidone and its five impurities. The initial development of the analytical method was based on the investigation of the retention behavior using mobile phases in the acidic pH range from 3 to 6 taken from the literature (5, 14, 15). It was observed that this pH range caused peak tailing of Impurities II and III. Initially, the mobile phase consisted of water, with the pH value adjusted with orthophosphoric acid. In order to improve peak symmetry for all compounds, the influence of phosphate buffer (25, 50, and 100 mM) and the acetate buffer (50 and 100 mM) as modifiers of the ionic strength, and sodium octane sulfonate as an ion-pairing reagent, were investigated. The optimal resolution, elution time, and peak shapes were achieved using 50 mM phosphate buffer with the pH value adjusted to 2.5. When acetonitrile was used as the organic modifier, the peaks were sharp and well separated, and had significantly shorter elution times compared to the results using methanol. Due to this advantage, acetonitrile was chosen as the organic modifier. Initially, a linear gradient program was used, since the impurities differ in polarity. However, it was found that for the separation of the critical pair, i.e., ziprasidone and Impurity II, application of isocratic elution within the time range from 6 to 10 min was necessary. Because TEA can mask free silanols and in that way prevent peak tailing from secondary interactions with stationary phase, its addition improved the peak shapes and resolution of the critical pair. It also reduced the elution time of the last eluted compound (Impurity V). According to the literature (14, 21), an earlier detection
Figure 4. Response surface plot of the response variables resolution between Impurity II and ziprasidone.
Figure 5. Response surface plot of the response variables resolution between ziprasidone and Impurity III.
of ziprasidone was carried out at 229 ± 1 nm. In this study, 250 nm was chosen for monitoring of the separation. At this wavelength, all compounds of interest absorb well, and no interference with the matrix was observed. Moreover, the proposed analytical procedure exhibits lower baseline noise compared to the method using 229 nm. The dependence of the chromatographic behavior of all of the studied compounds on temperature was studied. Retention decreased with a temperature increase, but room temperature (25°C) was considered optimal, due to the longer life of the column at this temperature than at higher temperatures.
718 Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011 Table 3. Accuracy of the HPLC method expressed as the recovery of the analyte spiked into pharmaceutical dosage form (capsule) Actual concentration, μg/mL
Compound Ziprasidone
Impurity I
Impurity II
Impurity III
Impurity IV
Impurity V
Amount found, μg/mL
Recovery, %
80
80.71
100
99.72
120
121.56
100.9 99.72
0.85
0.7
0.9
98.47
3.77
98.78
2.60
104.96
1.64
2.248
92.15
0.74
2.831
92.82
1.63
3.410
93.18
0.49
1.62
1.616
99.77
3.76
2.025
2.039
100.69
0.85
2.431
2.453
100.90
0.46
1.636
1.551
94.8
1.62
2.045
2.068
101.13
1.01
2.454
2.453
99.95
0.29
1.645
1.592
96.78
1.18
2.056
2.061
100.22
0.76
2.467
2.442
98.98
2.64
1.636
1.611
2.045
2.020
2.454
2.576
2.44
3.05
3.66
Under the optimal established chromatographic conditions, the obtained chromatographic parameters for ziprasidone and its five impurities were satisfactory (peak asymmetry, 0.8 0.8 indicates that the model fits well to the data (28). For “Rs Imp2-Zipr” and “Rs Zipr-Imp3,” the obtained responses were 0.8188 and 0.8939, respectively, which proves the goodness of the model and the design. At the same time, the Q2 values (representing the fraction of the variation predicted by
the model) for “Rs Imp2-Zipr” and “Rs Zipr-Imp3” were 0.7359 and 0.6415, respectively. This shows the high predictive ability of the model, since Q2 ≥ 0.5 indicates the validity of the model's prediction (28). To show the significance of different variables, VIPs were displayed in plots as bars, and the confidence intervals at the 95% confidence level as error lines (Figure 3). The variable is considered insignificant when VIP < 1 and the error line crosses zero. Upon examination of the plots, the variables, pH, KH2PO4 concentration, KH2PO4 concentration × KH2PO4 concentration, pH × pH, pH × KH2PO4 concentration, T × KH2PO4 concentration, and TEA% × KH2PO4 concentration were found insignificant and removed from the models. TEA concentration and column temperature were significantly effective. The increase of the column temperature was accompanied by an increase in resolutions, while an increase of TEA concentration resulted in an increase of “Rs Imp2-Zipr” and decrease of “Rs Zipr-Imp3” (Figures 4 and 5). Thus, from the surface plots it is evident that there is an optimum TEA concentration (1%) for both resolution responses (Figures 4 and 5), and the
720 Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011
y = –6.53 + 76.30x (r = 0.9993) y = –3.55 + 38.89x (r = 0.9997) y = –1.65 + 58.22x (r = 0.9999) y = 0.03 + 54.20x (r = 0.9993)
Figure 7. Chromatograms obtained by the gradient RP-HPLC method for determination of ziprasidone contained in two commercial formulations: (a) Zeldox powder for solution for injection, and (b) Zeldox capsules.
working temperature of 25°C was selected due to longer life of the column. There are certain advantages of the newly developed method over the one previously published (21). For example, the retention times of ziprasidone and the most common Impurities I and IV are twice as short in the new method. Further, the method was validated for its selectivity, accuracy, linearity, precision, LOD, and LOQ. Method Validation After establishing the optimal conditions for the separation, validation of the analytical method was performed in order to demonstrate that the new procedure is suitable for its intended purpose and that it meets proper standards of accuracy and reliability (29). The linearity of the analytical procedure was estimated within the range of 70–130 µg/mL for ziprasidone, and from the respective LOQ values to 6.0 µg/mL for the five main impurities. Statistical analysis of the obtained data was performed by linear regression using the least squares method. The obtained regression equations were as follows: y = 41.58 + 13.63x (r = 0.9991) y = –0.02 + 22.21x (r = 0.9999)
where x is the concentration in µg/mL and y is the peak area for ziprasidone and for Impurities I–V, respectively. As the obtained correlation coefficient (r) values for ziprasidone and all investigated impurities were higher than 0.999, it could be concluded that the calibration curves were within the acceptance criteria of linearity. In order to assess the accuracy of the method for each investigated compound, preanalyzed samples of capsules were spiked with a known amount of impurities and ziprasidone. The accuracy of the assay was evaluated in triplicate at each of three different concentration levels (80, 100, and 120%). The recovery values were calculated from the obtained calibration curve, and the accuracy results are presented in Table 3. The recovery range and RSD for all of the impurities were 92.15– 104.96% and 0.29–3.77%, respectively. No interference of the excipients with the peaks of interest was observed; hence, the proposed method is suitable for the quantitative determination of ziprasidone and its five impurities in the pharmaceutical dosage forms (Figure 6). The LOQ and LOD were experimentally determined based on the S/N approach. S/N of 10:1 and 3:1 were determined for each impurity. Evaluations were performed by injecting a series of the diluted solutions with known concentrations. The LOQ values for Impurities I–V were found to be 0.06, 0.06, 0.08, 0.07, and 0.04 µg/mL, respectively, which are equivalent to the impurity levels of 0.006, 0.006, 0.008, 0.007, and 0.004% calculated against the content of ziprasidone, respectively. The LOD values for Impurities I–V were equal to 0.016, 0.017, 0.024, 0.020, and 0.012 µg/mL, respectively (equivalent to the impurity levels of 0.0016, 0.0017, 0.0024, 0.002, and 0.0012% calculated against the content of ziprasidone, respectively). The HPLC– DAD gradient mode determination of the residual alkylating agent 5-(2-chloro-ethyl)-6-chlorooxindol (Impurity IV) in ziprasidone was reported earlier by Singh et al. (30). In that study, the LOQ and LOD values were 0.06 and 0.02%, respectively. The LOQ and LOD values of the currently proposed method 0.007 and 0.002%, respectively, show that this method is significantly more sensitive than the earlier one. Also, according to the regulatory framework for the potentially genotoxic impurities (31), the threshold of toxicological concern (TTC value) for Impurity IV is 0.004% for ziprasidone powder for the solution for injection. Because the LOD of this impurity is less than
Pavlovic et al.: Journal of AOAC International Vol. 94, No. 3, 2011 721
Table 6. HPLC assay of ziprasidone and its five impurities in the pharmaceutical dosage forms Impurity, % ± RSD, %b Sample
Ziprasidone content, %, ± RSD, %a
Zeldox capsule
99.8 ± 1.2
Zeldox, powder for solution for injection
100.3 ± 1.1
Impurity I
Impurity II
0.001 ± 3.24 0.005 ± 2.93 ND
ND
Impurity III
Impurity IV
Impurity V
c
ND
0.002 ± 2.65
ND
ND
0.007 ± 1.78
ND
a
Related to the label declaration.
b
Related to the content of ziprasidone.
c
ND = Not detected.
the TTC value, the maximum acceptable level of this compound could be detected with the proposed method. The repeatability of the devised method was checked by replicate sample injections (n = 5) of the six individual preparations of ziprasidone (100 µg/mL) and ziprasidone spiked with 0.2% of each impurity, except for Impurity II (added at 0.3%), with respect to the concentration of the target analyte ziprasidone (1 mg/mL). SD and RSD were calculated, and the values are given in Table 4. During this precision study, it was shown that the RSD value for ziprasidone was equal to 0.22%, and the RSD values for Impurities I–V were 0.23, 0.52, 0.94, 0.68, and 0.93%, respectively. These data confirm the high precision of the method. Robustness of the proposed method was tested by deliberate variations of several parameters, i.e., the buffer pH value, concentration of potassium hydrogen phosphate as the buffer component of the applied mobile phase, alterations in the flow rate, column temperature, and percent TEA added (32). From the data summarized in Table 5, it can be concluded that the proposed analytical method remained basically unaffected by the aforementioned deliberate changes in the analytical conditions. The developed method was applied to pharmaceutical formulations that contained ziprasidone. Figure 7 illustrates the separation of the impurities in the formulations tested. The results are presented in Table 6. Compared with the label declarations, the recoveries of ziprasidone from the dosage forms were high. The results obtained for the impurities in the Zeldox capsule and powder for the solution for injection met the limit requirements of manufacturer. Impurity IV, a potential genotoxin, was not detected in any batch tested in this study. An unknown impurity was detected at the retention time (tR) of about 14.7 min in the chromatogram of the sample of Zeldox powder for the solution for injection. Conclusions An RP-HPLC method was developed, validated, and applied for determination of the impurity levels and ziprasidone active ingredient in the capsules and in
the powder for the solution for injection. As there are no references in the literature concerning quantitative analysis of ziprasidone and its five impurities, the proposed method can be considered novel. The newly elaborated method was validated and found to be selective, sensitive, precise, accurate, and robust. Thus, it can be used for simultaneous identification and quantification of ziprasidone and its five impurities in the bulk drug substance and in dosage formulations. Furthermore, the impurity with the potential structural alert (IV) could be separated and detected using the developed method. Acknowledgments This work was supported by the Ministry of Science and Technological Development of the Republic of Serbia, Contract No. 172033. References (1) Greenberg, W.M., & Citrome L. (2007) CNS Drug Rev. 13, 137–177 (2) Rosa, A.R., Franco, C., Torrent, C., Comes, M., Cruz, N., Horga, G., Benabarre, A., & Vieta, E. (2008) CNS Neurosci. Therapeut. 14, 278–286 (3) Harrison, T.S., & Scott, L.J. (2006) CNS Drugs 20, 1027–1052 (4) Janiszewski, J.S., Fouda, H.G., & Cole, R.O. (1995) J. Chromatogr. B 668, 133–139 (5) Zhang, G., Terry, A.V. Jr, & Bartlett, M.G. (2007) J. Chromatogr. B 856, 20–28 (6) Suckow, R.F., Fein, M., Correll, C.U., & Cooper, T.B. (2004) J. Chromatogr. B 799, 201–208 (7) Aravagiri, M., Marder, S.R., & Pollock, B. (2007) J. Chromatogr. B 847, 237–244 (8) Janiszewski, J., Schneider, P., Hoffmaster, K., Swyden, M., Wells, D., & Fouda, H. (1997) Rapid Commun. Mass Spectrom. 11, 1033–1037 (9) Al-Dirbashi, O.Y., Aboul-Enein, H.Y., Al-Odaib, A., Jacob, M., & Rashed, M.S. (2006) Biomed. Chromatogr. 20, 365–368 (10) Zhang, G., Terry, A.V., Jr, & Bartlett, M.G. (2007) Rapid Commun. Mass Spectrom. 21, 920–928 (11) Zhang, G., Terry, A.V., Jr, & Bartlett, M.G. (2008) Biomed. Chromatogr. 22, 770–778
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