Development and validation of a microwave-assisted digestion technique as a rapid sample preparation method for the estimation of selenium in pharmaceutical dosage forms by ICP-OES

1. Introduction

The element selenium, CAS No. 7782-49-2, is a member of the group VIA of the periodic table and possesses metallic and nonmetallic characteristics ( 1 ). Selenium belongs to class 2B of elements according to the International Council for Harmonization Q3D guideline ( 2 ). These class-2B elements are less likely to be included in the therapeutic product due to their limited availability and poor ability to co-isolate with other materials. Therefore, they might not be considered in the risk assessment unless they are consciously incorporated while producing drug compounds, excipients, or other components of the drug product ( 2 ). Selenium sulfide is an anti-infective agent with antifungal and antibacterial properties and is used to cure dandruff and seborrheic dermatitis ( 3 ); hence, it is used as a dermatological shampoo.

Literature survey shows that selenium can be determined using different techniques, such as voltammetric analysis ( 4 , 5 ), spectrophotometry ( 6 , 7 ), chromatography (high-performance liquid chromatography [HPLC]) ( 8 , 9 ), atomic absorption spectroscopy ( 10 - 14 ), atomic fluorescence spectroscopy (AFS) ( 15 - 17 ), inductively coupled plasma optical emission spectroscopy (ICP-OES) ( 18 ), inductively coupled plasma mass spectrometry (ICP-MS) ( 19 ), HPLC combined with ICP-MS ( 20 ), and HPLC in conjunction with AFS ( 21 ). Most of the above-mentioned analytical techniques were found to be time-consuming and/or required high levels of skills. Inductively coupled plasma optical emission spectroscopy is the preferred technique as it is cost-effective for single-element analysis and is sensitive and reliable. On the other hand, the open digestion technique is dangerous, time-consuming, and inconsistent. Open digestion sample preparation followed by a non-specific titration was used for estimating selenium sulfide drug substance ( 22 ) and selenium sulfide topical suspension ( 23 ). This research work aimed to develop and validate a simple, cost-effective, robust ICP-OES method for estimating selenium in selenium sulfide drug substance and shampoo formulation using closed microwave digestion.

2. Materials and Methods

2.1. Chemicals, reagents, standards, and samples

Selenium sulfide drug substance was purchased from ABCR GMBH (Germany). Selsun blue dandruff shampoo with menthol, 1% selenium sulfide (Manufacturer: Sanofi), and selenium sulfide topical suspension USP, 2.5% lotion (Manufacturer: Perrigo) were bought from the open market. Selenium 10 mg/ml was acquired from Inorganic Ventures. All the chemicals used were of analytical reagent grade. Ammonium hydrogen difluoride was bought from Sigma-Aldrich (USA). Boric acid 99.99% was obtained from Alfa Aesar (USA). Concentrated nitric acid (70%) trace metal grade was acquired from Fisher (USA).

2.2. Instruments and equipment:

ICP-OES made of Thermo-scientific iCAP 6000 series with Helium/Kinetic energy discrimination mode was utilized. The microwave digestion system CEM MARS 6 with Easy Prep Plus Vessels was used. The Mettler Toledo analytical balance with model: ME204E was employed. A hot plate made by Royal Scientific (model RSW 127) was used.

2.3. Sample preparation:

The first phase in the technique development process involved the investigation of solubility. The method development trail was initiated by dissolving approximately 0.1 g of drug substance (i.e., selenium sulfide) in 100 ml of water. Similarly, 0.1 g of shampoo and topical suspension were dissolved in 100 ml of water. As an alternate method, closed microwave digestion with concentrated high-pure acids was considered for sample preparation. Closed vessel microwave digestion was initiated by using a varied ratio of acids. The trial observations are tabulated in table 1. All the sample matrices were subjected to microwave-assisted closed vessel digestion under controlled pressure and temperature.

Sample preparation optimization

Selenium sulfide drug substance

For the drug substance, 0.5 g of selenium sulfide was accurately weighed. Approximately 0.7 g of ammonium hydrogen difluoride was added to each vessel, followed by 1 ml of water. The samples were pre-digested for approximately 5 min in the ammonium hydrogen difluoride solution. A volume of 10 ml of concentrated nitric acid was added to each digestion vessel, and the samples were continued to pre-digest for around 15 min. The microwave reaction vessels were assembled, and the samples were digested according to the parameters described in the digestion step 1 (Table 3). The samples were allowed to cool before adding 10 ml of 4% boric acid required to complex the remaining hydrofluoric acid. The microwave reaction vessels were reassembled, and the samples were digested according to the parameters described in the digestion step 2 (Table 3). Digested samples were quantitatively transferred to individual 10 ml volumetric flasks. Samples were allowed to cool to room temperature before diluting to volume with water.

Selenium sulfide drug formulation

Samples were prepared by shaking the product container vigorously and mixing a portion of the shaken product in a beaker to ensure sample homogeneity. A volume of 0.5 g of Selsun blue dandruff shampoo and 0.2 g of selenium sulfide topical suspension USP were accurately weighed separately. Using a micropipette pipette, samples were dispensed into tared microwave vessels, and the product weight was recorded. Approximately 0.7 g of ammonium hydrogen difluoride was added to each vessel, followed by 1 ml of water. The samples were pre-digested for approximately 5 min in the ammonium hydrogen difluoride solution. Subsequently, 10 ml of concentrated nitric acid was added to each digestion vessel, and the samples were continued to pre-digest for around 15 min. The microwave reaction vessels were assembled, and the samples were digested according to the parameters described in the digestion step 1 (Table 3). The samples were allowed to cool before adding 10 ml of 4% boric acid required to complex the remaining hydrofluoric acid. The microwave reaction vessels were reassembled, and the samples were digested according to the parameters described in the digestion step 2 (Table 3). Digested samples were quantitatively transferred to individual 100 ml volumetric flasks. Samples were allowed to cool to room temperature before diluting to volume with water.

2.4. Rinsing solution for ICP-OES instrument autosampler - 2% nitric acid (v/v):

Slowly, 20 ml of nitric acid (concentrated) was added to 500 ml of water. This solution was diluted to 1 L with water and mixed well.

2.5. Preparation of 4% boric acid (w/v):

Approximately 40 g of boric acid was weighed and added to 1,000 ml of water. The mixture was stirred while gently heated until it completely dissolved.

2.6. Preparation of standard solutions:

Concentrated nitric acid, 4% boric acid, and 10 mg/ml selenium (Se) reference standard were transferred in 500 ml and 200 ml volumetric flasks accordingly (as volumes indicated in Tables 2 and 3) and diluted to volume with water to prepare calibration standards and calibration check standards.

2.7. Instrumental parameters for analysis

ICP-OES conditions

ICP-OES instrument was set up according to parameters listed in table 4 and calibrated with the calibration standards (10, 25, 50, 75, and 150 μg/ml) each time before use. Selenium (Se) concentrations were quantified using external calibration. Initial calibration verification was performed with the calibration check standard (50 μg/ml). Samples were bracketed with the calibration check standard following every sixth sample. The permissible cutoff for quality control (QC) verification was 5.0% of the claimed QC concentration.

2.8. Validation parameters

2.8.1. System suitability/linearity

Before initiating every validation parameter, all standard aliquots and blank solutions were injected in ICP-OES. The acceptance criterion for relative standard deviation (%RSD) of 5 replicate analyses of the 50 μg/ml selenium calibration checked standard was NMT 2.0%. The calibration check standard was injected regularly to ensure the proper running of ICP-OES. The linearity of the method was proven using the standard aliquot solutions. The correlation coefficient of the 5-point calibration curve was calculated, and the %RSD of the calibration check standard was monitored.

2.8.2. Specificity parameter

Selenium determination was performed on the most sensitive emission line at 196.090 nm. Thus, the specificity was defined as no spectral interferences exhibited at 196. 090 nm, and microwave vessels used for digestion demonstrated no sample carryover. Spectral interferences were evaluated by aspirating blank, calibration standard, and sample solutions into the ICP-OES as well as by collecting full frame profiles of the charge injection device chip in the detector. Method blanks were digested and analyzed to monitor selenium carryover in the microwave vessels.

2.8.3. Method precision

Six samples of Selsun selenium sulfide drug substance were prepared by weighing approximately 0.5 g of drug substance into microwave vessels and digesting according to the procedure. To prepare six samples of Selsun blue dandruff shampoo (SeS₂, 1%) with menthol, about 0.5 g of product was weighed into microwave vessels and digested according to the procedure. In addition, six samples of selenium sulfide topical suspension USP (SeS₂ 2.5%) lotion samples were created through the process of weighing around 0.2 g of product and digesting it according to the same procedure. Selenium concentrations were determined via ICP-OES analysis.

2.8.4. Intermediate precision

Similar to the method precision parameter, six samples of selenium sulfide drug substance, Selsun blue dandruff shampoo (SeS₂ 1%) with menthol and selenium sulfide topical suspension USP (SeS₂ 2.5%) lotion samples were prepared and analyzed in ICP-OES on a different day.

2.8.5. Accuracy

Accuracy was performed at three concentrations (each triplicate preparation) ranging from 10 ppm, 50 ppm, and 150 ppm for drug substance and drug product for selenium content. The % recovery in each level was calculated.

2.8.6. Limit of detection and limit of quantitation

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated from linearity solutions. The LOD for selenium was calculated from the linearity curve. Similarly, LOQ was determined.

LOD (µg/ml (ppm)) = 3.3 × Standard deviation/slope

LOQ (µg/ml (ppm)) = 10 × Standard deviation/slope

2.8.7. Robustness

Variations in instrument conditions (Table 5) were performed to evaluate robustness. The highest applied RF power setting for our ICP-OES instrument was 1,350 W. Therefore, the original work plan of 1,500 W was modified to test up to 1,300 W for the robustness study.

3. Results

3.1. Method developmental trials

During solubility studies, the selenium drug substance was found to be insoluble in water. The same result was confirmed in other studies ( 24 ). Further, Selsun blue dandruff shampoo with menthol and topical suspension USP was observed to be water-soluble.

To develop a common sample preparation method for selenium drug substance and drug product, the direct aqueous solution method for sample preparation was disregarded. Thus, the combination of microwave-assisted acid digestion and closed containers was used at higher temperatures without reagent and analyte losses and with a reduction in contamination risks.

In the closed vessel microwave-assisted digestion procedure, different volumes of concentrated nitric acid and hydrochloric acid had no significant impact on the digestion of the sample. Hence, according to Mathew et al. (2017) ( 25 ), molten ammonium hydrogen difluoride was utilized to digest the sample matrix. Ammonium hydrogen difluoride converted metal oxides to fluorides. Upon the addition of concentrated nitric acid, the fluorination reaction continued with the release of hydrofluoric acid (HF).

(NH₄F.HF) + HNO₃ NH₄NO₃ + 2 HF

Boric acid was required to complex the remaining hydrofluoric acid.

3.2. System suitability/linearity

The acceptance criterion for the correlation coefficient (r) of the 5-point calibration curve (10-150 μg/ml Se) was NLT 0.999. System suitability was achieved with a correlation coefficient value of 0.999980. (Figure 1 and Tables 6 and 7).

Figure 1. System suitability – linearity

3.3 Specificity

The specificity of the method was confirmed by measuring selenium at 196.090 nm, 203.985 nm, and 206.279 nm. Blank, 150 µg/ml calibration standard, 1% SeS₂ Selsun blue shampoo, and 2.5% SeS₂ Perrigo shampoo samples were measured at the above wavelengths. Hence in specificity solutions, spectral interferences were not observed at 196.090 nm, and selenium was not detected greater than 0.10 µg/ml in method blanks (Figure 2).

Figure 2. Specificity results when selenium was measured at 196.090 nm, 203.985 nm, and 206.279 nm

3.4 Method precision

The acceptance criterion for %RSD of the 6 samples each for selenium drug substance, Selsun blue dandruff shampoo (SeS₂ 1%) with menthol, and selenium sulfide topical suspension USP (SeS₂ 2.5%) was NMT 2.0%. For the drug substance, the %RSD for 6 replicate preparation was 0.2, for shampoo, for 6 replicate preparation, the %RSD was 1.7, and for the topical suspension, for 6 replicate preparation, the %RSD was 1.6 (Table 8).

3.5 Intermediate Precision

The results of 6 samples prepared and analyzed on different days were compared. The drug substance sample met current monograph specifications with an average selenium content of 99.2% for day 1 and an average of 99.8% for day 2. Both different days met the work plan criteria of NMT 2.0% RSD for 6 samples analyzed. The %RSD of overall precision for 12 samples was 0.6. The shampoo sample met current monograph specifications with an average selenium content of 93.8% for day 1 and an average of 93.6% for day 2. Both different days met the work plan criteria of NMT 2.0% RSD for 6 samples analyzed. The %RSD of overall precision for 12 samples was 1.4. The topical suspension sample met current monograph specifications with an average selenium content of 10.1.4% for day 1 and an average of 100.2% for day 2. Both different days met the work plan criteria of NMT 2.0% RSD for 6 samples analyzed. The %RSD of overall precision for 12 samples was 1.5 (Table 9).

3.6 Accuracy

Accuracy is the degree to which observed values and actual values agree. Drug substance recovery rates ranged from 98.5% to 100.7% on average. The average shampoo recoveries fell between 101.6% and 103.0%, which was comparable. For topical suspension, the range of typical recoveries was 99.7% to 101.7% (Table 10).

3.6 Limit of detection and limit of quantitation

The linearity curve was used to calculate the LOD and LOQ. The LOD values were 1.28 ppm and 3.89 ppm, respectively.

3.7 Robustness

System suitability results of all tested conditions (Figure 3) met acceptance criteria of NMT 2.0% RSD and NLT 0.999 for r.

Figure 3. Robustness result summary

4. Discussion

Selenium sulfide is commonly used in various dosage forms, such as shampoo and topical suspension, because of its antifungal and antibacterial properties. A common microwave-assisted digestion technique was developed and validated. The precision, specificity, linearity, accuracy, and robustness of the method for estimating selenium in selenium sulfide drug substances and various pharmaceutical dosage forms were demonstrated (Table 11). This newly developed microwave-assisted digestion technique has optimum sensitivity and is highly reproducible and time-saving than the existing methods (Table 12). This method can be applied to numerous matrices for a finished dosage of selenium sulfide formulations.

Acknowledgment

The authors are thankful to the management of GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India.

Authors' Contribution

Conceptualization, methodology and original draft preparation: A.K.P. Supervision, reviewing and editing: R.S.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Conflict of Interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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