Assessment of the Oxidative Degradation Pathways for Trifluoperazine Hydrochloride and Isopropamide Iodide Through a Validated Spe-hplc Methods and Application in Human Plasma
Ahmed S. Saad 1, Mona A. Mohamed 2*, Sara H. Koshek 2 and Mohamed R. El-Ghobashy1
1 Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr Elaini St., P.O. box 11562, Cairo, Egypt.
2 National organization of drug control and research (NODCAR), P.O. box 29, Giza, Egypt.
Trifluoperazine HCl (TFP) and isopropamide iodide (ISP) are commonly used as antipsychotic drugs. The stability of the two drugs was investigated against stress oxidation conditions and the effects of time and temperature on the degradation rate were studied. Separation was carried out on Agilent ZORBAX-CN column (150 X 4.6 mm id, 5µm particle size). The mobile phase composed of acetonitrile: 0.05 M phosphate buffer pH 3.5±0.1 containing 0.1% triethylamine adjusted with orthophosphoric acid 85 % 40:60 (v/v), pumped at flow rate 1 ml/min and UV detection was carried out at 220 nm.
The method had been validated as per the ICH guidelines and was successfully applied for the determination of the two drugs in their pharmaceutical dosage form in concentrations as low as 1 µg/ml. Human plasma were analyzed by including an early solid phase extraction step on a StrataTM -X-CW 33 µm weak polymeric cation exchanger prefilled tubes, conditions were optimized to maximize recovery of TFP and ISP and reduce noise in plasma samples.
Keywords: HPLC; Solid phase extraction; CN-column; Trifluoperazine Hydrochloride; Isopropamide Iodide.
Trifluoperazine hydrochloride (TFP) 10-[3-(4-Methyl-1-piperazinyl)propyl]-2-(trifluoromethyl)-10H-phenothiazine dihydrochloride, Figure 1a, is a neuroleptic phenothiazine used as an antidepressant commonly prescribed for treating psychiatric disorders and to relief schizophrenia symptoms. Isopropamide iodide (ISP), (4-Amino-4-oxo-3,3-diphenylbutyl)-methyl-di(propan-2-yl)azanium iodide, Figure 1b, is an anticholinergic with anti-anxiety and anti-spasmodic effect .
Literature survey indicated that the mixture of the two drugs was analyzed spectrophotometrically[2-6], and by using HPLC technique; however, neither of the mentioned methods dealt with the simultaneous determination of TFP and ISP in the presence of their degradation products in pharmaceutical dosage forms nor in biological samples.
The current work confirmed the relatively low stability of TFP under stress oxidation conditions, for instance, a thorough study was carried out to investigate different factors governing the oxidation process.
A stability indicating assay isocratic HPLC method was developed to monitor the drugs’ behavior under stress condition. The method was capable of simultaneous determination of the two drugs in the presence of TFP oxidative degradation product, Figure 1c . The developed method was applied to pharmaceutical formulation as well as to human plasma samples containing the two drugs. For biological application purpose, it was mandatory to develop a simple yet efficient sample preparation procedure by including a solid phase extraction (SPE) procedure prior to chromatographic separation. Conditions were optimized and the whole method was validated to meet the purpose of the study.
Figure1a, 1b, 1c: Drug Structures
Material and methods
Agilent 1100 series liquid chromatograph with quaternary pump (model G1311A), variable wavelength detector (model G1314A) and degasser model Agilent 1260 Infinity (model G1322A), data acquisition was performed using Agilent Chemstation PC user interface. Separation was performed on Agilent ZORBAX CN-column (150 X 4.6 mm id, 5µm particle size). Hanna instruments® pH-meter-Romania was used for pH adjustment of the buffer. Water purification for HPLC analysis was carried out using Purelab-flex water system-UK. The mobile phase was mixed and degassed using ultrasonic vibration (Crest® sonicator, USA) for 10 min and filtered using 0.2 µm membrane filter (Sartorius AG® membrane filter).
Degradation was carried in a boiling water bath (BŰCHI water bath B-480).Agilent 1200 series liquid chromatograph with quaternary pump (model G1311A), variable wavelength detector (model G1314B), diode array detector (model G1315D), manual injector (model G1328B) and degasser (model G1322A), data acquisition was performed using Agilent Chemstation PC user interface.
Reagents and Materials
Acetonitrile (MACRON, U.S), methanol (Fisher chemical, UK), triethylamine (Loba Chemie, India), orthophosphoric acid 85% (SHAM), and formic acid (Piochem) used, were of HPLC grade.
Analytical grade potassium dihydrogen orthophosphate (EL Nasr Pharmaceutical Chemicals Co.) and hydrogen peroxide (30% w/v) (Panreac, E.U.) were used. TFP and ISP standards were obtained from (Kahira pharmaceutical and chemical Industrial Co., Cairo, Egypt), purity was reported to be 100% and 99.4% for TFP and ISP, respectively. Stellamide® tablets, manufactured by (Kahira pharmaceutical and chemical Industrial Co., Cairo, Egypt). Batch number 1210776, each tablet contains 1.18 mg Trifluoperazine hydrochloride equivalent to 1 mg trifluoperazine base and 6.8 mg isopropamide iodide equivalent to 5 mg isopropamide base, was purchased from local market.
Chromatographic separation was performed on Agilent ZORBAX CN-column (150 X 4.6 mm id, 5µm particle size), as shown in Figure 2. A mobile phase consisting of acetonitrile: 0.05 M phosphate buffer (prepared by dissolving 6.8gm potassium dihydrogen orthophosphate in 900ml distilled water containing 1ml triethylamine and adjusted to pH 3.5±0.1 with orthophosphoric acid then diluted with distilled water to 1000 ml) with ratio 40:60 (v/v) and a flow rate of 1 ml/min, the column effluent was monitored at 220nm.
Figure 2: HPLC chromatogram of TFP (100 µg/ml, Rt 9.065 min) and ISP (100 µg/ml, Rt 4.740min) using the specified chromatographic conditions.
1.1. Standard solution
Working standard solutions 200 µg/ml of TFP and ISP were prepared separately, using the mobile phase as a solvent. Hydrogen peroxide (0.5 N) solution was prepared in distilled water.
1.2. Degradation procedure
TFP and ISP were subjected to forced degradation conditions. One mg of each drug dissolved in 2 mL 0.5 M aqueous hydrogen peroxide solution, left for 8 minutes in a boiling water bath. The solution was evaporated on a vacuum evaporator. The residue was transferred to 10-mL measuring flask using the mobile phase and the volume was completed using the same solvent, degradation was confirmed by the disappearance of TFP peak and the appearance of another peak at retention time 3.6 minutes as shown in Figure 3a and Figure 3b.
Figure 3a: HPLC chromatogram of ISP (100 µg/ml, Rt 4.908min) showing complete degradation of TFP (100 µg/ml, Rt 9.091min) and appearance of the main sulfoxide degradation product of TFP (Rt 3.261 min) using the specified chromatographic conditions.
Figure 3b: HPLC chromatogram of a synthetic mixture of TFP (100 µg/ml, Rt 9.091min) and ISP (100 µg/ml, Rt 4.738 min) in the presence of TFP oxidative degradate ( Rt 3.341 min) using the specified chromatographic conditions.
Effect of time
One mg of each drug dissolved in 2 mL 0.5 M aqueous hydrogen peroxide solution was subjected to reflux in 0.5 M hydrogen peroxide for different time intervals, while the temperature was held constant at 100oC. The study was carried out till complete degradation was achieved.
Effect of temperature
One mg of each drug dissolved in 2 mL 0.5 M aqueous hydrogen peroxide solution was subjected to reflux in 0.5 M hydrogen peroxide for 8 minutes at different temperatures.
Aliquots of TFP and ISP working standard solutions were separately transferred into a series of 10-ml measuring flasks and the volume was completed with the solvent to obtain a concentration range of 1-100 µg/ml. Twenty microliters of the prepared solutions were injected into the chromatograph under the previously described chromatographic conditions. Calibration graphs were constructed and regression equations were computed for each component.
Assay of pharmaceutical preparation
Five tablets of Stellamide® were accurately weighed then grinded using a mortar and a pestle. A weight equivalent to 2.95 mg TFP and 17 mg ISP were taken, 25 ml solvent were added and sonicated for 30 minutes, after filtration, 1ml of filtrate was transferred to 10-ml measuring flask and completed to final volume using mobile phase.
Quantification of TFP and ISP in human plasma
Standard solutions (0.05, 0.1, 0.2, 0.4, 0.8 mg/ml) of the two drugs were prepared in distilled water, 50 µl of the prepared solutions were transferred into test tubes each contains accurately 450 µl of human plasma, 2.5 ml acidified water pH 6 using formic acid was added to each tube, mixed using vortex mixer for 1 min., followed by solid phase extraction (SPE) procedures.
Solid phase extraction was carried out on StrataTM -X-CW 33 µm weak polymeric cation exchanger (60mg / 3ml, tubes), the stationary phase was conditioned using 1 ml methanol and equilibrated using 1 ml water then loaded with 3 ml of sample. The stationary phase was washed with 1 ml water followed by 1 ml methanol, the stationary phase was allowed to dry at 5 mmHg for 2 minutes, and the drugs were eluted using 2×0.5 ml 5% formic acid in methanol. The eluate was filtered using 0.22 µ membrane filter and 20 µl were injected into the chromatograph.
Results and discussion
High-performance liquid chromatography still represents the principal tool for separation and quantification of drugs in multicomponent preparations as well as in stability studies. The current work aims to develop sensitive and precise HPLC method preceded by a reproducible and efficient sample preparation technique for the simultaneous determination of TFP and ISP in the presence of their oxidative degradation products. The method was further adapted to adopt different matrices such as pharmaceutical dosage form and human plasma. TFP and ISP were subjected to forced oxidative degradation conditions, the whole degradation process was evaluated by purity assessment of the resulting peaks using diode array detector and the two intact drugs were selectively quantified as shown in Figure 4.
Figure 4: Purity assessment of ISP peak.
The two drugs were subjected to stress oxidation conditions at which isopropamide iodide proved to be stable; whereas, TFP suffered complete degradation after 8 minutes of treatment with 0.5 M aqueous hydrogen peroxide solution at 100oC, where TFP peak, at retention time (tR 9.901 minutes), vanished completely and another peak appeared at a relatively lower retention time (tR 3.3 minutes).
The effect of time and temperature on the degradation process of both drugs was closely monitored under forced degradation conditions. At constant temperature, 75.80% of trifluoperazine hydrochloride was degraded after 2 minutes and complete degradation occurred after 8 minutes, results are shown in Table 1. Whereas the effect of temperature was studied at constant time (8 minutes) and concentration of hydrogen peroxide, at which 76.70% of trifluoperazine hydrochloride was degraded at 70oC and complete degradation occurred at 100oC, results are shown in Table 2. On the other hand, isopropamide iodide manifested significant stability throughout the whole degradation process and up to 5 hours of the degradation process.
Optimum chromatographic separation was achieved on Agilent ZORBAX-CN column (150 X 4.6 mm id, 5µm particle size). Due to the relatively high polarity of ISP, the moderately polar cyano-column was selected to achieve proper retention of the drugs. Isocratic elution using a mobile phase consisting of acetonitrile: 0.05 M phosphate buffer containing 0.1% triethylamine and adjusted to pH 3.5±0.1 with orthophosphoric acid in the ratio 40:60 (v/v) resulted in adequate peak symmetry within reasonable run time at room temperature. Due to low absorptivity of ISP, the column effluent was monitored at 220 nm, where both, ISP and TFP, exhibits adequate sensitivity.
Table 1: Results of forced degradation of TFP at 100oC for different time intervals
|Time (min.)||Recovery % of TFP|
Table 2: Results of 8 minutes forced oxidative degradation of TFP at different temperatures
|Temperature (oC)||Recovery % of TFP|
|Room temperature (25±2 oC)||100.0 %|
|70 oC||23.3 %|
|80 oC||15.0 %|
|90 oC||7.3 %|
|100 oC||0.0 %|
In order to apply the developed method to a biological specimen such as human plasma, solid-phase extraction was employed for rapid and efficient sample cleanup to extend the life expectancy of the instrument and the analytical column. Reversed stationary phase (StrataTM –X) showed poor retention for both drugs, which could be attributed to their relatively polar chemical structure; on the other hand, strong cation exchangers would result in a strong irreversible retention of ISP, due to its strongly basic nature. Therefore, a reversed phase weak polymeric cation exchanger (StrataTM -X-CW 33 µm) was selected, which has the ability to retain non-polar basic compounds reversibly depending on the pH of the mobile phase.
The SPE procedure necessitates four successively steps to reach optimum matrix purification and drug recovery. Conditioning was performed using methanol and equilibration using water to acclimate the stationary phase for reversible retention of the basic and neutral drugs in the subsequent steps. Spiked human plasma specimens were treated with acidified water to impair protein binding, the prepared sample solutions were loaded onto the stationary phase; then, the loaded stationary phase was washed using water followed by methanol to remove matrix interference of acidic and neutral (phospholipids) impurities, respectively, without affecting the retention of the two cationic drugs that remain retained on the ionized cation exchanger, as confirmed by the absence of the two drugs’ peaks at their expected retention times in the wash, Figure 5 . Finally, the elution step was performed using acidic solution containing 5% formic acid in organic solvent, at which the stationary phase became unionized, and the drugs were eluted in methanol. Elution step was performed two successive times to ensure complete elution of the two drugs. The optimized SPE procedures effectively reduce the baseline noise caused by the biological sample matrix, as shown in Figure 6.
Figure 5: HPLC chromatogram of the wash under the specified chromatographic conditions.
Figure 6: HPLC chromatogram of TFP and ISP in human plasma using solid phase extraction under the specified chromatographic conditions.
Calibration graphs were constructed to assess linearity by relating the peak area to the corresponding analyte concentration and regression equations were calculated. The method was validated according to ICH guidelines as shown in Table 3. The proposed method successfully determined the two drugs in a pharmaceutical formulation, Table 4, and spiked human plasma, Table 5. The accuracy of the method was assessed by applying standard addition technique, results are represented in Table 6. Proper system suitability and performance of the method was ensured by comparing the calculated system suitability parameters to reference values, as shown in Table 7.
Table 3: Assay validation sheet of the proposed method.
|Linearity Range (µg/ml)||1-100||1-100|
|Correlation coefficient (r)||1||1|
|Intermediate precision (RSD%)(b)||0.51123||0.78221|
|Accuracy(%)±SD(c)||100.13 ± 0.41||100.06 ± 0.23|
Acetonitrile: Buffer (38: 62 by volume)
Acetonitrile: Buffer (42: 58 by volume)
100.40 ± 0.83
100.72 ± 0.55
100.55 ± 0.23
100.54 ± 1.70
(a) The interday (n = 3), RSD of three concentrations (10, 50 and 100μg/mL) for TFP and (10, 50 and 100μg/mL) for ISP repeated three times in three successive days.
(b) The intraday (n = 3), RSD of three concentrations (10, 50 and 100μg/mL) for TFP and (10, 50 and 100μg/mL) for ISP repeated three times within the day
(c) Accuracy (mean ± SD) assessed using a minimum of nine determinations over a minimum of three concentration levels covering the specified range.
(d) Robustness (mean ± SD) assessed by analysis of 10μg/mL TFP and 50μg/mL ISP using acetonitrile: buffer (38:62 by volume) repeated three times and acetonitrile: buffer (42:58 by volume).
Table 4: Application of the proposed method to pharmaceutical formulation.
|10||100.77 ±0.400||50||96.73 ±0.828|
(a) Average of three determinations.
Table 5: Determination of spiked TFP and ISP in human plasma
|Conc (µg/ml)||Recovery(a) %|
- Average of three determinations.
Table 6: Application of standard addition technique to the pharmaceutical preparation
|Taken (µg/ml)||Standard added (µg/ml)||Recovery(a) %||mean||SD||RSD %|
(a) Average of three determinations.
Table 7: System suitability testing parameters of the proposed HPLC method
|Parameters||TFP||ISP||Reference values (a)|
|tR (Retention time)||9.065||4.740||>1|
|N (Column efficiency)||3578||3931||N > 2000
Increases with efficiency
the efficiency of the separation
|K (retention factor)||5.34||2.79||1-10 acceptable|
|α (separation factor)||1.91||> 1|
to theoretical plates)
|0.0042||0.0038||The smaller the value, the higher the column efficiency|
|T (tailing factor)||1.348||1.305||T<2, T=1 for symmetric peak|
|Rs (Resolution)||9.50||Rs >2|
Values defined by FDA Center of Drug Evaluation and Research’s reviewer guidance on validation of chromatographic methods (November 1994)
A selective and sensitive HPLC method was developed to assess the effect of forced oxidative degradation on TFP and ISP. Although TFP was susceptible to oxidation within 8 minutes of exposure to hydrogen peroxide, ISP was exhibited significant stability. The developed HPLC method was able to determine TFP and ISP in pure powder form and in their pharmaceutical formulation. Biological application of the developed method necessitates the incorporation of a sample preparation procedure, where SPE technique was employed for effective matrix cleanup, stationary phase and conditions were meticulously selected to achieve minimal baseline noise and maximum recovery of the two drugs.
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