A New Stability Indicating Reverse Phase HPLC Method for the Simultaneous Determination of TPPO and TPP in the Presence of Phenylhydrazine Derivative in Penicillamine Drug Substance

Penicillamine is a conventional disease-modifying antirheumatic drug (DMARD) and is chemically identified as (2S)-2-Amino-3-methyl-3-sulfanylbutanoic acid (or) D-3-Mercaptovaline. The molecular formula is C5H11NO2S and the molecular weight is 149.21. The chemical structure of Penicillamine is shown in Figure 1. Penicillamine is a heavy metal chelator, used in the treatment of Wilson disease, rheumatoid arthritis, and cystinuria. Penicillamine is a good chelating agent and highly soluble in water, it is generally resistant to metabolic degradation. It has a molecular structure adapted to binding metals, and retains this chelating activity despite the pH of body fluids. Penicillamine forms complexes with heavy metals that are less toxic than the free metallic ion. Penicillamine chelates copper through stable complex formation by thiol groups and this allows increased renal copper excretion [1].

Figure 1 Chemical Structure of Penicillamine Drug Substance.

Impurities present in drug substances and drug products can impact the quality, safety and efficacy of the drug substance and drug product. Triphenylphosphine (TPP), triphenyl phosphine oxide (TPPO) and phenylhydrazine derivative are the potential impurities existing in the route of synthesis of Penicillamine. Triphenylphosphine is a crucial and commonly used chemical that acts as a reducing agent in organometallic synthesis. Triphenylphosphine and triphenylphosphine oxide decompose on heating and produce toxic fumes, phosphorus oxides and phosphine (phosphorus trihydride). Exposure to triphenylphosphine and triphenylphosphine oxide cause skin and serious eye irritation, and may cause respiratory irritation. Triphenylphosphine has low to moderate acute toxicity based on results from animal tests following acute oral exposure. In rats, the median lethal dose (LD50) by the oral route ranged from approximately 700 mg/kg bw (in olive oil) to > 6400 mg/kg bw (in aqueous suspension), depending on the vehicle used. In mice, the oral LD50 (in olive oil) was 1000 mg/kgbw. Toxic effects observed within 1-4 days of exposure included dyspnoea, apathy and impaired balance. The LD50 was reported to be approximately 700 mg/kg bw [2]. Esa et al. evaluated triphenylphosphine and triphenylphosphine oxide have been shown to suppress immune responses in vitro [3]. In marine, exposure of triphenylphosphine oxide activated natural immune genes and disrupted lipid metabolism in mussels. Short-term exposure did not cause oxidative stress in mussels. Chronic exposure results in significant oxidative damage and oxidative stress in mussels [4]. A lethal dose of triphenylphosphine oxide for animals (beagle dogs) is a single oral dose, either 300 or 500mg/kg in corn oil leads fatal to uncoordinated body movements [5].

In the literature, various techniques have been reported for the quantification of triphenylphosphine (TPP) and triphenyl phosphine oxide [6-13] as triphenylphosphine oxide. A method for the determination of triphenylphosphine based on its oxidation to triphenylphosphine oxide by iodine or chloramine-T in acid medium in the presence of benzene or carbon tetrachloride is described [6]. Spectrophotometric determination of triphenylphosphine by molibdate 510 nm as phosphorus [7]. An isocratic reversed phase high performance liquid chromatographic method is described for monitoring concentrations of triphenylphosphine (TPP) and other compounds involved in an oxygen-transfer reaction catalyzed by a molybdenum complex [8]. Determination of triphenylphosphine oxide (TPPO) in active pharmaceutical ingredients by HPLC [9]. A simple assay for lipid hydroperoxides based on triphenylphosphine oxidation and HPLC [10]. Determination of triphenylphosphine oxide for quantification of hydroperoxides in lipids by HPLC [11]. Determination of peroxide value of edible oils by FTIR spectroscopy using polyethylene film [12]. Measurement of peroxide values in oils by triphenylphosphine/ triphenylphosphine oxide assay coupled with FTIR-ATR spectroscopy comparison with idometric titration [13].  In above procedures triphenylphosphine is converted into triphenylphosphine oxide and triphenylphosphine oxide will be quantified.

So, it is important to develop a sensitive analytical method for the quantification of triphenylphosphine and triphenylphosphine oxide in drug substances.  The interest of the study is to quantify both triphenylphosphine and triphenylphosphine oxide in a single method. Triphenylphosphine is slowly converted into triphenylphosphine oxide in aqueous solutions and it is stable in organic solvents like Acetonitrile and Methanol. In this study, a stability indicating reverse phase HPLC method for the determination of triphenylphosphine, triphenylphosphine oxide and phenylhydrazine derivative impurities in Penicillamine drug substance has been developed, optimized and validated as per the ICH guidelines [14].

MATERIAL AND METHODS

Materials:

Penicillamine and its related substances were prepared in APL Research Centre-II (A Division of Aurobindo Pharma Ltd). Triphenylphosphine and triphenylphosphine oxide standards purchased from Sigma Aldrich. Ammonium dihydrogen phosphate, HPLC grade acetonitrile, HPLC grade methanol, hydrochloric acid, sodium hydroxide and hydrogen peroxide were purchased from Merck India. Milli-Q grade (Millipore, MA, USA) water was used during experiments. 0.45µm PVDF membrane filters were procured from Merck Life Science Pvt Ltd.

Instrumentation:

All experiments were carried out on a Waters alliance e2695 (Waters Corporation, Milford, MA, USA), separation module consisting of a binary pump, column oven, and an auto-injector equipped with 2489 UV/Visible detector. Peak purity was performed on 2998 photodiode array detector (PDA). Empower software was used for signal monitoring and data processing.  

Preparation of standard, sample and Buffer solutions:

The standard solution was prepared at a level of about 1 µg/mL each of Triphenylphosphine oxide, Phenylhydrazine derivative and Triphenylphosphine reference standards in diluent by diluting stock solution containing 0.25 mg/ml. Methanol is used as a diluent. A sample solution at about 2 mg/ml concentration is prepared diluent.

Buffer solution of 0.01M ammonium dihydrogen phosphate was prepared by dissolving 1.15 g/L of the respective salt in water. Buffer solution was filtered through 0.45 µm porosity membrane filter. Buffer solution is used as Mobile phase A. Acetonitrile is used as Mobile phase B.

RESULTS AND DISCUSSION

Method development:

The aim of the work is simultaneous determination of triphenylphosphine oxide, triphenylphosphine and phenylhydrazine derivative. The proposed analytical method should separate other related substances from analytes. Method development initiated by using a HPLC column consists of octadecylsilane (C18) as a stationary phase. Phosphoric acid and acetonitrile are used as mobile phase A and B with a gradient elution (T _min / % B - 0/50, 28/80, 33/80, 35/50, 45/50) at a flow rate of 1.0ml/min. In this phase columns Triphenylphosphine oxide and TPP are well separated. The triphenylphosphine oxide and phenylhydrazine derivatives have very less resolution in the octadecylsilane stationary phase. Octylsilane (C8) stationary phase used to separate the analytes. In this experiment, triphenylphosphine oxide and phenylhydrazine derivatives have less resolution. A column having sub-3-micron particle size with core technology is used to improve the resolution between triphenylphosphine oxide and phenylhydrazine derivatives and reduce the back pressure. Mobile phase A is changed to ammonium dihydrogen orthophosphate at a concentration of 0.01M is used to improve the resolution. Chromatographic parameters are optimized to get optimal resolution between triphenylphosphine oxide and phenylhydrazine derivatives and avoid interferences from sample matrices. The resolution between triphenylphosphine oxide and phenylhydrazine derivative is set as system suitability criteria for the proposed analytical method and the proposed method has been validated as per the ICH guidelines.

Detection Method:

Separation of analytes is achieved on an Ascentis Express C8 column with dimensions of         150mm x 4.6 mm and 2.7 µm. Analytes are eluted through gradient elution by using 0.01M ammonium dihydrogen phosphate buffer and Acetonitrile are used as Mobile phase A and Mobile phase B respectively. The proposed gradient programme for the separation is                               (T _min / % of Mobile phase-B) 0/40, 10/70, 25/70, 27/40, 35/40 at a flow rate of 0.8ml/min. The analytical column was thermostated at a temperature of 20°C and the analytes were monitored at 210 nm by injecting 10 µL of sample solution.  The retention times of triphenylphosphine oxide, phenylhydrazine derivative and Triphenylphosphine are about 5.6, 6.0 and 17minutes respectively and molecular structures are shown in figure 2.

Triphenylphosphine oxide

Phenylhydrazine derivative

Triphenylphosphine

Figure 2 Chemical Structures of analytes.

METHOD VALIDATIONS

The proposed method has been validated as per ICH guidelines to prove its competence. The validation parameters studied were specificity, sensitivity, linearity, precision (Repeatability and Intermediate precision), accuracy, and robustness. The results obtained from the experiments are summarized in the next paragraphs.

System suitability:

The standard solution at a level of about 1 µg/mL each of Triphenylphosphine oxide, Phenylhydrazine derivative and Triphenylphosphine is injected into the HPLC system for system suitability. The resolution between “Triphenylphosphine oxide and Phenylhydrazine derivative” peaks is a minimum of 2. The column efficiency is determined from the phenylhydrazine derivative peak is not less than 12000 USP plate counts and USP tailing for the same peak is not more than 1.5. Representative chromatogram of standard solution is shown in figure 3.

Figure 3 Representative HPLC chromatogram of Standard – Triphenylphosphine oxide, Phenylhydrazine derivative and Triphenylphosphine.

The RSD for the respective peak areas of six injections of the standard solution is not more than 5.0%. The results are tabulated in Table 1.

Specificity stands for the ability of the method to judge the analytes accurately in the presence of other components present in the sample matrix. The specificity and stability indicating nature of the proposed method is performed in the presence of forced degradation. The wet and dried forced degradation studies of penicillamine were subjected to acid, base, peroxide, thermal, photolytic, and humidity conditions. Stress studies were performed at a test concentration level for the penicillamine drug substance. Conditions were tabulated in Table 2. Stressed samples were analyzed using the PDA detector to evaluate the ability of the proposed method to separate the degradation products from analytes. Triphenylphosphine oxide, phenylhydrazine derivative and triphenylphosphine were not formed during all the stress conditions. This implies that the proposed method was specific.

Representative chromatograms of Triphenyl phosphine oxide, Phenylhydrazine derivative and   Triphenyl phosphine is shown in figure 4 - 6.

Figure 4 Representative HPLC chromatograms of Triphenylphosphine oxide.

Figure 5 Representative HPLC chromatograms of Phenylhydrazine derivative.

Figure 6 Representative HPLC chromatograms of Triphenylphosphine.

Limit of Detection and Limit of Quantification:

Limit of detection (LOD) and limit of quantification (LOQ) are the key parameters which reveal the sensitivity of an analytical method. The LOD and LOQ concentrations were estimated for triphenylphosphine oxide, phenylhydrazine derivative and triphenylphosphine by using the slop of the linearity curve, ranging from 5 to 150% of the specification level. Predicted concentrations of LOD and LOQ solutions for analytes were prepared and injected into HPLC as per the methodology. The % RSD values for the precision of analytes ranged from 0.9 – 2.3 and 0.4 – 3.5 for LOD and LOQ, respectively. From these results, it was concluded that the proposed method had adequate sensitivity for the detection and quantification of triphenylphosphine oxide, phenylhydrazine derivative and triphenylphosphine in Penicillamine drug substance. The results are tabulated in Table 3.

Linearity:

The linearity of an analytical method will determine the range of that proposed method, in which the results are directly proportional to the concentration of the analyte in the sample.  Linearity was performed by injecting ten diluted solutions ranging from LOQ to 150% of the specification level. The linearity was evaluated by linear regression analysis, i.e., y = mx + c, where y is the peak area, x is the concentration, and m and c values represented the slope and intercept, respectively. The correlation coefficients for triphenylphosphine oxide, phenylhydrazine derivative and triphenylphosphine from the linear graph were found to be 0.9999, 0.9999 and 0.9990 respectively.  The results are tabulated in Table 4.

Accuracy was demonstrated by the standard addition method, in which known quantities of triphenylphosphine oxide, phenylhydrazine derivative and triphenylphosphine were added to the penicillamine sample solution. Spiked solutions were prepared in triplicate at the LOQ level and 50 – 150 % w/w level. The analytes of penicillamine were determined from the spiked sample solutions and the percent recovery was calculated, and it was found to lie in between 95.1 and 103.7 %w/w. The statistical analysis showed that the method was accurate. The results are tabulated in Table 5.

Representative chromatograms of spiked sample, sample and diluents are shown in figure 4 - 6.

Figure 7 Representative HPLC chromatograms of Penicillamine drug substance - Spiked with Triphenylphosphine oxide, Phenylhydrazine derivative and Triphenylphosphine.

Figure 8 Representative HPLC chromatograms of Penicillamine drug substance –As such sample.

Figure 9 Representative HPLC chromatograms of Diluent

Precision:

The system precision has been demonstrated by injecting six replicate injections of standard solution into the HPLC system and the %RSD of area response for six replicate measurements was found to be less than 5.

Method precision has been evaluated by means of repeatability and intermediate precision. To determine the repeatability and intermediate precision of the method, replicated (n=6) injections of the spiked sample at specification level of analytes of penicillamine were carried out and precision was expressed as % RSD. The results are tabulated in Table 6.

Robustness of the method indicates the ability of the method to remain unchanged even after variations in critical method parameters. The effect of variation in flow, percentage organic gradient composition, column oven temperature, and detection wavelength on system suitability criteria was studied. The resolutions between triphenylphosphine oxide and phenylhydrazine derivative were found to be more than 3.0 in each variation, which indicates the robustness of the method. The statistical analysis of robustness data was illustrated in Table 7.