Development and validation of a simple and rapid ICP-OES method for quantification of elemental impurities in voriconazole drug substance

The aim of the present study was to develop and validate an inductively coupled plasma optical emission spectroscopic (ICP–OES) method for quantification of elemental impurities, i.e., Lead, Palladium, and Zinc, in voriconazole drug substance, and this method was employed for the regular sample analysis of Lead, Palladium, and Zinc in voriconazole drug substance for pharmaceutical use. The method has been validated using RF power of 1150 W, auxiliary gas of 0.5 L/min, and nebulizer flow of 0.4 L/min and plasma view at axial mode for Lead and Palladium and radial mode for Zinc. The wavelength was monitored for Lead, Palladium, and Zinc at 220.3 nm, 340.4 nm, and 213.8 nm respectively. The method is selective and is capable of detecting desired elemental impurities with regulatory acceptance limits in the presence of other elements. The validation experiments involve the demonstration of system suitability, specificity, LOD and LOQ, linearity, precision, and accuracy experiments. The linearity results obtained ˃ 0.9990 for all three impurities. The proposed method is simple, sensitive quality control tool for the simultaneous quantitative determination of Lead, Palladium, and Zinc at low levels in voriconazole drug substance.


Background
Voriconazole is a second-generation triazole antifungal agent used to treat serious fungal infections. Chemically, voriconazole is (αR,βS)-α-(2,4-difluorophenyl)-5-fluoro-βmethyl-α-(1H-1,2,4-triazol-1-ylmethyl)-4-pyrimidineethanol (OR) (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-YL)-1-(1H-1,2,4-triazol-1-YL) butan-2-OL with a molecular formula of C 16 H 14 F 3 N 5 O and a molecular weight of 349.3 [1]. Voriconazole was first synthesized by Pfizer in the brand name of Vfend® which has broad-spectrum activity against pathogenic yeasts, including Aspergillums, Cryptococcus, and Candida species and ineffective against Rhizopus, Zygomycosis reduced activity [2,3]. Pharmaceutically, voriconazole formulations are available in both oral and intravenous administration. The similar drug of Fluconazole structurally modified to develop the Voriconazole [4]. In the literature, a few analytical methods have been reported for the quantitative estimation of voriconazole in pharmaceutical dosage form and in biological fluids [5][6][7][8], electrophoresis and chromatographic separation of voriconazole stereoisomers [9], determination of voriconazole impurities by HPLC [10], and determination of voriconazole in formulation by HPLC [11]. Quantitative determination of voriconazole in rat and beagle dog plasma reported [12]. In voriconazole, synthetic process Lead, Palladium, and Zinc-related complex molecules are used as catalytic agents. During the synthesis of drug substances, elemental impurities may arise from several sources and may be catalysts that were added intentionally in synthesis or may be present as impurities. These elemental impurities do not provide any therapeutic benefit to the patient, and levels in the drugs should be controlled within acceptable limits. Depending upon their permitted daily exposures, lower levels have to be considered for elemental risk [13]. The intake of palladium causes fever, hemolysis, erythema, edema, and eye irritation [14]. Lead causes depression, loss of appetite, intermittent abdominal pain, nausea, diarrhea, constipation, muscle pain, and problems with sleep [15]. Zinc causes nausea, vomiting, loss of appetite, stomach cramps, diarrhea, and headaches [16]. Hence, residual elemental impurities controls are required in voriconazole drug substance for regulatory requirements. Several colorimetric and spectrometric methods for determination of Lead [17][18][19], Palladium [20][21][22][23], and Zinc [24][25][26][27][28] was reported. In view of this, we have developed and validated the inductively coupled plasma optical emission spectroscopy (ICP-OES) method and presented in this research paper. To the best of our knowledge, no method for the determination of Palladium, Lead, and Zinc by ICP-OES was reported in voriconazole drug substance. The developed method was validated according to International Conference on Harmonization (ICH) (Q2A) guidelines [29]. The chemical structure of voriconazole is shown in Fig. 1.

Chemicals and reagents
Sulfuric acid, hydrochloric acid, and hydrogen peroxide purchased were from Merck chemicals limited. ICP standards, Lead, Zinc, and Palladium each 1000 mg/L and multi-element standard solution IV (1000 mg/L) were purchased from Sigma-Aldrich. Voriconazole working standard was gifted from APL Research Centre-II (a division of Aurobindo Pharma Limited) and Milli Q-water produced from Milli pore water system. Borosil type glassware and calibrated 100-1000 μl pipettes were used.

Equipment
An inductively coupled plasma system equipped with optical emission spectrophotometer make Thermo Fisher Scientific with data handling system controlled with iTEVA software used for method development and validation.

Preparation of solutions
Lead and Palladium standard stock solution (Pb: 5 ppm and Pd: 10 ppm) Fifty microliters of Lead ICP standard solution (1000 mg/L) and 100 μl of Palladium ICP standard solution (1000 mg/L) was transferred into a 10 ml clean dry volumetric flask, mixed, and made up to volume with Milli-Q Water.
Preparation of calibration standard-1 (Lead: 0.015 ppm, Palladium: 0.03 ppm, and Zinc: 3.9 ppm) Seventy-five microliters of Lead and Palladium standard stock solution and 97.5 μl of Zinc ICP standards (1000 mg/L) pipette into a 25 ml clean dry volumetric flask, containing 15 ml of water, 2.5 ml of Hydrogen peroxide solution, 1.0 ml of Hydrochloric acid, and slowly 0.5 ml of sulfuric acid was added. The solution was and diluted to the mark with Milli-Q water.
Calibration standard-2 (Lead: 0.03 ppm, Palladium: 0.06 ppm, and Zinc: 7.8 ppm) One hundred fifty microliters of Lead and Palladium standard stock solution and 195 μl of Zinc ICP standards (1000 mg/L) were mixed into a 25 ml clean dry  volumetric flask, containing 15 ml of water, 2.5 ml of hydrogen peroxide solution, 1.0 ml of hydrochloric acid, and slowly 0.5 ml of sulfuric acid was added. The solution was mixed and diluted to the mark with Milli-Q water.
Calibration standard-3 (Lead: 0.06 ppm, Palladium: 0.12 ppm, and Zinc: 15.6 ppm) Three hundred microliters of Lead and Palladium standard stock solution and 390 μl of Zinc ICP standards (1000 mg/L) were mixed into a 25 ml clean, dry volumetric flask, containing 15 ml of water, 2.5 ml of hydrogen peroxide solution, 1.0 ml of hydrochloric acid, and slowly 0.5 ml of sulfuric acid was added. The solution was mixed and diluted to the mark with Milli-Q water.

Blank solution
Transferring 5.0 ml of hydrogen peroxide solution into a 50 ml clean dry volumetric flask, containing 30 ml of water, add 2.0 ml of hydrochloric acid and slowly 1.0 ml of sulfuric acid was added. The solution was mixed and diluted to the mark with Milli-Q water.

Sample solution
Accurately weighed and transferred about 0.6 g of sample in to 10 ml clean dry volumetric flask, 1.0 ml of hydrogen peroxide solution, 0.4 ml of hydrochloric acid was added, and sonicate to dissolve. Then, 0.2 ml of sulfuric acid was slowly added, mixed well, and make up to volume with Milli-Q water. (To obtain the clear solutions, centrifuge the sample solutions at 6000 rpm for 10

Method validation Specificity
To demonstrate specificity of test sample (control sample) and test sample spiked with multi elements, including Lead, Palladium, and Zinc standard (spiked sample) in triplicate were aspirated as per ICP-OES test methodology and determine the Lead, Palladium, and Zinc contents. Acceptance criteria is % difference between mean of each content in control sample and spiked sample should be not more than10.0% as per method validation protocol. The specificity results are tabulated below (Table 1).

LOD and LOQ
Limit of detection (LOD) was obtained by aspirating a linear series of Lead 0.005 to 0.06 ppm, Palladium 0.01 to 0.12 ppm, and Zinc 1.0 to 15.6 ppm standard solutions were prepared in seven concentration ranges       respectively. From the linearity data, slope and residual sum of squares (STEYX) obtained from above LOD and limit of quantitation (LOQ) values were predicted. And acceptance criteria is %RSD is not more than 33.0 for LOD and 10.0 for LOQ. LOD was calculated as 0.08 ppm for Lead, 0.2 ppm for palladium, and 5.0 ppm for Zinc. LOQ was obtained as 0.25 ppm for Lead, 0.5 ppm for palladium, and 16.7 ppm for Zinc. % RSD for 6 replicates obtained for LOQ was 3.6%, 0.7%, and 0.4% for Lead, Palladium, and Zinc respectively. Summarized results are shown in Tables 2, 3, 4, 5, and 6.

Linearity
Analytical method linearity is defined as the ability for showing the response of the analyte is proportional to the analyte concentration within a given range. The peak response obtained from the ICP-OES was plotted against corresponding concentrations to obtain the calibration graph. The results of the linearity study gave linear relationship over the concentration range of 50% to 200% for Lead and Palladium, and of 12.8% to 200% for Zinc of specification level. From the regression analysis, the coefficient of determination R 2 was 0.9990 for all three analytes, indicating a linear relationship between the concentration of analyte and response under the peak. Linearity results for three analytes are shown in Table  7 and linearity graphs shown in Figs. 2, 3, and 4.

Accuracy (recovery)
Accuracy expresses the familiarity of conformity between the value obtained and the value which is accepted either as a predictable true value or an accepted reference value. The accuracy of the method is determined by recovery studied for Lead, Palladium, and Zinc. The sample solutions were prepared in triplicate by spiking Lead, Palladium, and Zinc at LOQ level, 50%, 100%, and 150% of specification and analyzed individually as per test method. The percentage recovery of added Lead, Palladium, and Zinc and the % RSD were calculated for each replicate samples. Acceptance criteria is recovery should be between 70.0 and 150.0%. Accuracy results tabulated below (Table 8).

Precision (ruggedness)
The closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under given conditions. The precision was checked both for system and method. System precision was demonstrated by aspirating standard solution of Lead, Palladium, and Zinc at LOQ level and measured six times. The requirement of acceptance criteria is %RSD should not be more than 10.0.The results were within the acceptable criteria. The experimental system precision results are shown in Table 9.

Method precision
Method precision was determined by preparing six sample solutions individually using single batch of voriconazole drug substance spiked with Lead, Palladium, and Zinc at LOQ level and aspirated into ICP-OES as per methodology. Ruggedness was obtained by six sample solutions were prepared individually using single batch of voriconazole (used for method precision) spiked with Lead, Palladium, and Zinc at LOQ level and aspirated into ICP-OES as per methodology and determined Lead, Palladium, and Zinc contents using different analyst on different day. Acceptance criteria is % RSD should not be more than 10.0% for method precision, ruggedness, and overall. Results were given in

Discussion
The research work focused on simple and rugged ICP-OES method development and validation [30-34] of three elemental impurities, i.e., Pb, Pd, and Zn, in voriconazole drug substance simultaneously. For the analysis of voriconazole, drug substance sample digestion was done using 2.5 ml of hydrogen peroxide solution, 1.0 ml of hydrochloric acid, and slowly 0.5 ml of sulfuric acid. Among the three elemental impurities determined in voriconazole drug substance samples, one elemental impurity is class 1 (Pb), one impurity is class 2B (Pd), and one impurity is class 4 (Zn) according to the elemental impurities classification based on toxicity from ICH guidelines [13]. Till date, no ICP-OES method reported for the simultaneous determination of above three elemental impurities in any drug substance. So effort was through to develop simple, rapid ICP-OES method, and it was validated with LOD and LOQ 0.08 μg/g, 0.2 μg/g, 5 μg/g, and 0.25 μg/g, 0.5 μg/g, 16.7 μg/g for Pb, Pd, and Zn respectively. Linearity obtained was 0.9995, 0.9990, and 0.9997 for Pb, Pd, and Zn respectively. The average recovery found was 110% for Pb, 87.4% for Pd, and 98.6% for Zn. Determined concentrations of these analytes in drug substance samples were lower than the limits established by the chapter 232 [34]. In true sense, the daily maximum dose for voriconazole is 200 mg and thus the risk is very little; therefore, the limits established considering this maximum daily dose are even elevated (Table 13).

Conclusion
ICP-OES method was developed and validated according to current ICH and FDA guidelines to quantify Lead, Palladium, and Zinc in voriconazole drug substance. The proposed ICP-OES method has been evaluated over the linearity, precision, accuracy, and specificity and proved convenient and effective for the quality control of voriconazole drug substance. Thus, the present study demonstrates that ICP-OES has the advantages over other conventional analytical methods for the determination of Lead, Palladium, and Zinc because of sensitivity, i.e., the lower limit of detection, for Lead, Palladium, and Zinc in voriconazole drug substance. Therefore, the method can easily be adopted for routine quantitative analysis of Lead, Palladium, and Zinc present as residual impurities in voriconazole drug substance.