Determination of elemental impurities of Arsenic, Cadmium, Mercury, Lead and Palladium content in Testosterone propionate by using ICP-MS

 

P. Suresh¹*, Konda Ravi Kumar²

¹Vishwa Bharathi College of Pharmaceutical Sciences, Perecharla-522009, A.P., India.

²Department of Pharmaceutical Chemistry, Hindu college of Pharmacy, Guntur, India.

 

ABSTRACT:

The aim of the present work is to develop and establish a validated analytical method for the determination of arsenic, cadmium, mercury, lead and palladium content in testosterone propionate by using inductive coupled plasma mass spectroscopy (ICP-MS). Samples were analyzed after a preparation of sample solution by dissolving in suitable solvents of concentrated nitric acid and concentrated hydrochloric acid. In the present method, RF power of 1550 watts, RF matching is 1.80 V, nebulizer flow of 0.10 rps and plasma view at spectrum mode were used. Octopole conditions are He flow is on, He flow rate is 4.3 mL/min and energy discrimination is 3.0 V were used. Significant savings in sample volumes, reagents, analysis cost and time are realized. Arsenic, cadmium, mercury, lead and palladium are primary concerned due to their high toxicity and potential contaminants should be limited in testosterone propionate and the developed method was validated according to ICH and USP guidelines. The correlation coefficient, recovery rate, LOD and LOQ reached the acceptable limits. The validated method was selective, sensitive, rapid and capable of the determination of elemental impurities of arsenic, cadmium, mercury, lead and palladium content in bulk drugs.

 

 

 

INTRODUCTION:

Testosterone is one of the important male gonadal hormones and is essential for the development of male sex organs and secondary sexual characteristics in men. Low level of Testosterone in serum occurs due to inadequate function of testis which results in male hypogonadism^1,2. The symptom of hypogonadism includes erectile dysfunction, decrease bone density, decrease volume of ejaculate and increase body fat^3-5.

 

Various analytical techniques were design for the estimation of inorganic metal impurities include titration, ion-exchange chromatography, capillary electrophoresis and spectroscopic techniques like flame photometry, fluorimetry, atomic absorption spectroscopy and inductive coupled plasma. Among the above-mentioned techniques, inductive coupled plasma is an important tool for the detection and quantification of elements in accurate manner and it works on the basis of atomic spectrometry. Inductive coupled plasma also further includes 2 types which are ICP-optical emission spectroscopy and ICP-mass spectroscopy. Inductively coupled plasma mass spectrometry (ICP-MS) is a robust and widely used technique for multi-elemental and isotopic analysis of environmental materials that has shown clear advantages when compared with other analytical techniques such as inductively coupled plasma atomic emission spectrometry (ICP-AES), flame atomic absorption spectrometry (F-AAS), and electro thermal atomic absorption spectrometry (ET-AAS). The basic setup for ICP-MS analysis requires the sample introduction as a liquid solution and thus, for solid matrices, an acid digestion procedure becomes mandatory⁶. Literature revealed that few methods like estimation of metals in fruits⁷, blood and urine⁸, Germanium oxide⁹ and pharmaceutical excipients are reported.

 

Various research articles are reported for the development and analysis of testosterone esters^10-12. Method for estimation of impurities in testosterone formulations using HPLC was reported by Bharti et al.,¹³.

 

This research paper describes the methodology and validation procedure by ICP-MS for the determination of elemental impurities of arsenic, cadmium, mercury, lead and palladium content in testosterone propionate and the method was validated according to ICH (Q2R1), USP 232 and 233 guidelines^14-17.

 

MATERIALS AND METHODS:

Chemical and laboratory reagents:

Trace metal grade concentrated hydrochloric acid (Sigma Aldrich Chemical), Trace metal grade concentrated nitric acid (Sigma Aldrich Chemical), multi-element standards of Cd, Pb, As, Hg (IV-Stock- 41 from Inorganic Ventures), Pd standard (Merk). Test materials were stored at the recommended storage conditions provided on the supplier’s certificate of analysis. All solutions prepared from the test materials were stored at room temperature. All analytical chemicals sourced by Almac Sciences were of trace analysis grade or equivalent. All volumetric flasks used in preparations were polymethyl pentene (PMP), polypropylene (PP) Class A or equivalent quality polymer. All sample preparation steps and measurements at Almac Sciences- Mass Spectrometry laboratory (FDA and MHRA certified) were carried out under GMP.

 

Preparation of solutions:

Preparation of diluent:

Transferred 20 mL of Con. HNO₃ and 10 mL of Con. HCl into a 1000 mL volumetric flask, the flask contains 500 mL of Milli-Q water and further diluted the flask up to the volume with Milli-Q water and mixed well.

 

Preparation of standard solutions:

Preparation of 100 ppm of Hg and Pd standard solution:

Pipetted out 1.000 mL of each 1000 ppm of Hg and Pd Standard solution into two different 10 mL volumetric flasks and diluted each flask make up to the mark with diluent and mixed well.

 

Preparation of 10 ppm of As, Cd and Pb standard solution:

Pipetted out 0.100 mL of each 1000 ppm of As, Cd and Pb Standard solution into three different 10 mL volumetric flasks and diluted each flask make up to the mark with diluent mixed well.

 

Preparation of standard mix solution:

The above solutions are diluted to obtain 0.150 ppm of As, 0.050 ppm of Cd and Pb, 0.300 ppm of Hg, 1.000 ppm of Pd. Taken all the above concentrations into 50 mL volumetric flask and made up the mark with diluent and mixed well.

 

Preparation of calibration standard solutions:

Preparation of calibration blank solution (Level- 1):

Diluent is used as a calibration blank solution.

Preparation of calibration standard solution (Level-2 to 7):

Pipetted out 0.100 mL, 0.200 mL, 0.400 mL, 0.600 mL, 0.800 mL and 1.000 mL of standard mix solution into six different 10 mL volumetric flasks and diluted each flask made up to the mark with diluent and mixed well.

 

Preparation of standard check solution:

Pipetted out 0.400 mL of standard mix solution into a 10 mL volumetric flask and diluted the flask made up to the mark with diluent and mixed well.

 

Preparation of calibration standard solutions using above standard mix solution:

The solutions for calibration standards are prepared as per the dilutions mentioned in table 1.

 

Table 1: Preparation of calibration standard solutions using above standard mix solution

 

Preparation of sample solution:

Homogenized the sample and weighed 0.1 g of sample into a clean and dry microwave digestion vessel and added 4.0 mL of Con. HNO_3, 2.0 mL of Con. HCL and then kept for pre-digestion for 10 minutes for reaction and closed vessel and kept in microwave digestion and use followed the digestion program as 800 W power, Ramp for 15 min at 190ºC for 20 min holding time. After completion of microwave digestion, allowed the sample solution to cool at room temperature and transferred the solution into a 25 mL volumetric flask and diluted up to the mark with milli-Q-water and mixed well.

 

Preparation of sample blank solution:

Prepared as per sample preparation without sample and follow the procedure described under preparation of sample solution.

 

INSTRUMENTATION:

An Agilent 7800 ICP-MS was used for all measurements. The instrument was equipped with standard nickel sampling and skimmer cones, a glass concentric nebulizer, quartz spray chamber and quartz torch with 2.5 mm id injector. The instrument also features a fourth-generation collision/reaction cell, the ORS4. The ORS4 includes a standard helium (He) mode cell gas line which provides effective removal of most common polyatomic interferences (He gas flow is approximately 4.2 mL/min). He mode provides sensitivity for the detection of elements at the levels defined in the regulatory guidelines. An Agilent ASX-520 auto-sampler was used to deliver the samples. The standard solution of 1 ppb mixture of Li, Y, Tl tuning solution was used with expected m/z values at 7, 89 and 205 with % RSD NMT 15%.

 

METHOD VALIDATION:

System precision:

To demonstrate the system precision, aspirated the standard preparation six times and evaluated the system precision by computing the relative standard deviation of the response of Arsenic (As), Cadmium (Cd), Mercury (Hg), Lead (Pb) and Palladium (Pd).

 

Preparation of Calibration standard for precision:

Pipetted 0.800 mL of standard mix solution into a 20 mL volumetric flask anddiluted the flask made up to the volume with diluent and mixed well.

 

Acceptance Criteria for System Precision:

% RSD for the CPS response of six replicate aspirations of 100 % level standard solution preparation for each analyte should not be more than 20.

Specificity:

It was assessed to show the absence of isobaric and polyatomic interference via quantifying the spiked samples against the calibration standard solutions and meeting the accuracy requirements.

 

Preparation of Calibration standard for specificity:

Pipetted 0.100 mL of standard mix solution into a 10 mL volumetric flask and diluted the flask made up to the volume with diluent and mixed well.

 

Preparation of calibration blank solution for specificity:

Used diluent as a calibration blank solution for specificity

 

Acceptance criteria for specificity:

The significant interference of calibration blank at the mass of each analyte should be lower than calibration standard solution.

 

Limit of detection (LOD) and Limit of Quantification (LOQ):

Limit of detection is determined by aspirating appropriate number of diluted standards using regression plot of residual against linearity concentration of diluted standard.

 

Preparation of Sample blank solution:

Transferred 4.0 mL of Con. HNO3 and2.0 mL of Con. HCl into a cleaned and dried microwave digestion vessel, then kept pre digestion for 10 minutes and closed the vessel and kept in microwave digestion and followed as per sample preparation procedure.

 

LOD Preparation:

Taken 3.3 mL of LOQ Solution into a 10 mL volumetric flask and made up to the volume with diluent. This solution was considered as LOD solution.

LOD solution is considered as 33% of LOQ level.

 

Acceptance criteria for LOD and LOQ:

The response of each analyte at LOD level should be significant above the blank response. LOQ for each analyte should not be more than 50% of the specification limit.

 

Observation at LOD and LOQ precision:

The verification of LOD and precision of LOQ study at LOD and LOQ level concentration. Verified LOD level on visual detection basis and evaluated the precision at LOQ concentration by computing % RSD of area observed in LOQ preparation.

 

Acceptance Criteria for LOQ Precision and observation at LOD:

The response for each analyte at LOD level should be sufficiently higher than the response of calibration blank. Response.

% RSD of each analyte at LOQ level should not be more than 20.

 

Linearity:

To demonstrate the linearity of analytical method over the range of minimum LOQ to 250% of specification level concentration. Aspirated each level of calibration standard and evaluated the correlation coefficient.

 

Preparation of Linearity calibration standards (Level-1 to 6) using standard mix solution:

The solutions for calibration standards are prepared as per the dilutions mentioned in table 2.

 

Table 2: Preparation of Linearity calibration standards (Level-1 to 6) using standard mix solution

 

Acceptance criteria for Linearity:

Correlation coefficient for each analyte should not be less than 0.99.

 

Method precision:

Demonstrated method precision by preparing six sample preparations as per methodology and evaluate the method precision by computing the relative standard deviation for the residue of each analyte. Aspirated the calibration blank, standard solutions and six sample preparations into ICP-MS.

 

Preparation of sample solution (Pre-1 to Pre-6) (As such sample):

Weighed 0.1010 g, 0.1014 g, 0.1012 g, 0.1015 g, 0.1010 g and 0.1014 g of sample into a cleaned and dried six different microwave digestion vessels and added 4.0 mL of Con.HNO₃,2.0 mL of Con. HCl then kept pre digestion for 10 minutes and closed vessels and kept in microwave digestion and remaining procedure followed as sample preparation.

 

Acceptance criteria for Method Precision:

The % RSD for content of each analyte from the six preparation of the sample/spike at specification level solutions should not be more than 20.

 

Preparation of 100% spiked sample solution (Pre-1 to Pre-6) (100% Spiked at sample):

Weighed 0.1014g, 0.1012g, 0.1016g, 0.1011g, 0.1012g and 0.1015 g of sample into six different cleaned and dried microwave digestion vessels and each vessel added 1.000 mL of standard mix solution, 4.0 mL of Con. HNO₃, 2.0 mL of Con. HCl. Then kept pre digestion for 10 minutes for reaction and closed vessels and kept in microwave digestion and remaining procedure followed as sample preparation.

 

Acceptance criteria for Method Precision (Spiked):

The content of % RSD of six spiked sample preparation for each analyte should not be more than 20.

 

Accuracy:

Demonstrated the accuracy of the analytical method, prepare test sample in triplicate as per methodology, prepare recovery samples by spiking known amount of each analyte (at level of LOQ, 50%, 100% and 150% of specification level concentration) to test sample in triplicate at each level.

 

Preparation of sample at LOQ level spiked solution (Pre-1 to 3):

Weighed 0.1014g, 0.1015g and 0.1010g of sample into a cleaned and dried three different microwave digestion vessels and each vessel added0.250 mL of standard mix solution, then added 4.0 mL of Con. HNO3, 2.0 mL of Con. HCl. Then kept pre digestion for 10 minutes for reaction and closed vessels and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Preparation of sample at 50% level spiked solution (Pre-1 to 3):

Weighed 0.1011g, 0.1013g and 0.1015g of sample into a cleaned and dried three different microwave digestion vessel and each vessel added 0.500 mL of standard mix solution, then added 4.0 mL of Con. HNO3, 2.0 mL of Con. Hydrochloric acid. Then kept pre digestion for 10 minutes for reaction and closed vessels and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Preparation of sample at 100% level spiked solution (Pre-1 to 3):

Weighed 0.1015g, 0.1010g and 0.1012g of sample into a cleaned and dried three different microwave digestion vessel and each vessel added 1.000 mL of standard mix solution, then added 4.0 mL of Con. HNO3, 2.0 mL of Con. Hydrochloric acid. Then kept pre digestion for 10 minutes reaction and closed vessels and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Preparation of sample at 150% level spiked solution (Pre-1 to 3):

Weighed 0.1010g, 0.1014g and 0.1016g of sample into cleaned and dried three different microwave digestion vessels and each vessel added 1.500 mL of standard mix solution, then added 4.0 mL of Con. HNO3, 2.0 mL of Con. Hydrochloric acid. Then kept pre digestion for 10 minutes for reaction and closed vessels and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Acceptance criteria for accuracy:

The mean % recovery for each analyte at each level should be 70 to 150.

 

Range:

Based on the linearity, precision and accuracy studies the range of method As, Cd, Hg, Pb and Pd content in Testosterone Propionate by ICP-MS. LOQ to 250% of the specification level.

 

Intermediate Precision:

The intermediate precision of the method is determined by the prepared six spiked samples are analyzed by the proposed method by different analyst. The result will be shown in the tables indicate that the method is Rugged. This study is performed in different day by different analyst. Evaluated the intermediate precision by computing the relative standard deviation for the response of As, Cd, Hg, Pb and Pd. Aspirated the calibration blank, standard solutions and six spiked sample preparations into ICP-MS.

 

Acceptance criteria for intermediate precision:

% RSD for the response of six sample spiked preparation of each analyst should not be more than 20.

The cumulative (Method precision and Intermediate precision) %RSD content of each analyte should not be more than 25.

 

Robustness:

Robustness of analytical method shall be established by demonstrating its reliability against deliberate changes in sample preparation.

 

Preparation of sample at 100% level spiked solution (Ideal):

Weighed 0.1014g of sample into a cleaned and dried microwave digestion vessel and added 1.000 mL of standard mix solution, then added 4.0 mL of Con. HNO3,2.0mL of Con. Hydrochloric acid. Then kept pre digestion for 10 minutes for reaction and closed vessel and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Preparation of sample at 100% level spiked solution (+10% HNO3, HCl):

Weighed 0.1010g of sample into a cleaned and dried microwave digestion vessel and added 1.000 mL of standard mix solution, then added 4.4 mL of Con. HNO3,2.2 mL of Con. Hydrochloric acid. Then kept pre digestion for 10 minutes for reaction and closed vessel and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Preparation of sample at 100% level spiked solution (-10%HNO3, HCl):

Weighed 0.1015g of sample into a cleaned and dried microwave digestion vessel and added 1.000 mL of standard mix solution, then added 3.6 mL of Con. HNO3, 1.8 mL of Con. Hydrochloric acid. Then kept pre digestion for 10 minutes for reaction and closed vessel and kept in microwave digestion. After completion of microwave digestion program followed the same procedure described under sample solution preparation.

 

Acceptance criteria for robustness:

The % of recovery for each analyte in spiked sample preparation should be 70 to 150.

 

RESULT AND DISCUSSION:

The main objective of the study was to develop a suitable ICP-OES method to quantify palladium content present in testosterone propionate. During the method development, palladium standard prepared of any working concentration was monitored at different possible emission lines of 338.57, 340.46 and 345.85 nm by aspirating the solution. Among these emission lines at applied target RF power of 1500 W the response for palladium was evaluated. The response of palladium was prominent at 340.46 nm. Moreover, the baseline was to be good at above stated wavelengths compared to other emission lines of elements. The possible inferences were not observed at this emission lines. The method was optimized at these wavelengths during method development. To get better sensitivity plasma view mode was selected as axial. As this mode is much larger than that viewed by normal radial or normal size on ICP resulting higher intensity for elements.

 

System Precision:

The % RSD was found to be within the specification (% RSD should not be more than 5 %). The system precision was checked by analyzing six replicates of standard solution (100 % level). The concentration of each element for each replication was calculated by using the standards (Tables 3).

 

Table 3: System Precision Results

 

Specificity:

Prepared one spiked sample (sample spiked with 100% standard solution) as per the method and analyzed. The obtained results are reported in Table-4. All the results are well within the acceptance criteria and results indicate that the method is specific.

Table 4: Specificity Results

 

Table 5: LOD and LOQ results

 

Table 6: Observation of LOD and LOQ Precision results

 

Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ are calculated as per the method mentioned. LOD and LOQ levels are found and the details are furnished in table 5.

 

Observation of LOD and LOQ precision:

The verification of LOQ was done at LOD and the results are shown in table 6.

 

Linearity:

Linearity was evaluated by preparing a linear series of standard solutions of elements in the concentrations range from LOQ level to 250 % level of specification level. From the linear solutions, correlation co-efficient, Y intercept, slope was calculated and thus the linear relationship of concentration versus counts was verified over the range specified. The correlation coefficient was found to be greater than 0.9999 which indicates good linearity. The results are reported in table 7.

 

Table 7: Linearity results for each element

 

Method precision:

Method precision was performed using spiked solutions as well as without spiking the samples. The obtained results are well within the limits. They are tabulated in tables 8 and 9.

 

Table 8: System suitabilityresults

 

Table 9: % RSD response of six spiked sample preparation

 

 

Table 10: % Recovery for each element

 

 

 

Table 11: % Recovery values of robustness parameters

All the parameters results were well within the acceptance criteria.

 

 

Accuracy:

Sample solutions were spiked with palladium at LOQ level, 50%, 100 % level and 150 % level with respect to sample concentration. Each spiked solution was prepared in triplicate and aspirated. The recovery of palladium was calculated against the standard working concentration (Table-8)

 

Robustness:

The recovery levels tested in preparations varying the % of nitric acids. The recovery values were found to be comparable. The % of recovery for each analyte in spiked sample preparation is in between 70 to 150 (table 11).

 

CONCLUSION:

A validated and accurate ICP-MS method has been developed to estimate elemental impurities content in testosterone propionate. The method is selective and is capable to quantitate impurities in the presence of other trace elements. The method has been validated in terms of specificity, precision, linearity, accuracy and limit of detection and quantification. The validated method is selective, precise, accurate and robust. This method can be used to estimate elements not only in testosterone propionate but also in other drug substances.

 

 

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Received on 19.11.2020          Modified on 30.12.2020

Accepted on 25.02.2021          ©AJRC All right reserved