INTRODUCTION:

With increasing environmental concerns, the application of the guiding principles of green pharmaceutical chemistry has gained increased importance. In this context, despite its heavy metal status, bismuth containing compounds are considered non-toxic and safe which is primarily attributed to their insolubility in neutral aqueous solutions such as biological fluids, relatively air moisture stable and easy to handle¹. Due to these reasons, over the years, several bismuth-based compounds have been widely used in different areas of life e.g., semiconductors, cosmetic products (such as pigments in eye shadow, lipsticks and hair dyes) and in medicines².

Compounds containing bismuth are widely used in medical applications for more than three centuries. It is an active ingredient of various pharmaceutical products. Bismuth compounds remain important components of stomach remedies, such as Pepto-Bismol (bismuth subsalicylate, BSS), De-Nol (colloidal bismuth subcitrate, CBS) and bismuth subnitrate and some of these are available as over-the-counter treatments^1,3. It acts as a mucosal protective agent and is able to prevent the surface of ulcers from eroding as a result of stomach acidity and proteinase. Due to its good antibacterial properties, bismuth-containing pharmaceuticals are most commonly used in the eradication of Helicobacter pylori, the causative agent for diseases like gastritis, peptic ulcer and even gastric cancer^1,4.

The need for the analysis of elements in pharmaceuticals is becoming increasingly more important, both from the perspective of product quality and patient safety. The products intended for human consumption must be characterized as completely as possible⁵. The careless use of bismuth-containing pharmaceuticals can result in encephalopathy, renal failure and other adverse effects^3,4. Hence, the accurate determination of bismuth in bismuth-containing pharmaceuticals gives an assurance of the actual amount present in the medicine that is consumed by the patient. It helps the consumer know whether the composition labelled on the drug blister pack or bottle is as per the prescription, in relation to safety and efficacy of the medicine.

The determination of bismuth in any solid sample requires bismuth in the solid matrix to be brought into solution prior to its analysis by solution based-instrumental techniques. The analytical challenges associated with sample matrix depend on the selection of sample preparation method which is key to the success of an analysis. Open-vessel hot plate digestion and open/closed-vessel microwave assisted digestion techniques are routinely used methods for the digestion of various solid matrices ranging from environmental, biological, geological and pharmaceutical prior to instrumental detection^6-8. Different sample preparation methodologies based on conventional hot-plate or microwave digestion has been described in the literature for the determination of Bi in pharmaceutical products containing bismuth. A majority of these methods report the use of conventional hot plate for sample digestion^9-18, using large volumes of concentrated acids and take long periods for sample dissolution. In contrast closed-vessel microwave digestion offers decomposition of both inorganic and organic matrices under controlled conditions of temperature, pressure and time. It has gained widespread acceptance as an effective method for sample preparation, reducing the digestion times and reagent amounts, can be operated at higher temperatures of 250 - 300°C, avoiding contamination and loss of volatile species, and improving safety¹⁹.

Nowadays, one of the most important trends in solid sample preparation for elemental analysis is the search for environment friendly processes. According to “Green Chemistry” recommendations, the use of dilute acids for digestion leads to relevant advantages such as reduction of acid amount, production of smaller amounts of residues, reduction of process blank values and avoids the risk of explosion^20-22. Taking these advantages into consideration, a number of recent applications demonstrated the potential of dilute nitric acid solutions in microwave-assisted digestion of wide variety of samples of different origins e.g., food^23,24, medicinal plants²⁵, biological²⁶ and botanical materials^27,28. Recently this approach was successfully applied for the determination of trace elemental impurities in biodiesel²⁹, sports supplements³⁰ and pharmaceutical samples³¹.

The present work aimed to develop a MWAD method using dilute nitric acid solution for the digestion of solid pharmaceutical samples, for the determination of bismuth by HG-AFS. The microwave digestion parameters were optimized and the efficiency of digestion was evaluated based on the percentage recovery of bismuth and residual carbon content in the digested sample solution. Accuracy was evaluated by comparing the values obtained with a reported method using conventional hot plate digestion and with the value given by the manufacturer. To the best of our knowledge till date a similar study is not reported in open literature on the determination of bismuth in pharmaceuticals.

MATERIALS AND METHODS:

Instrumentation:

All the microwave digestions have been performed in a Monowave 300 microwave reactor with 30mL glass vials (Anton Paar GmbH, Austria). Temperature can be controlled via built-in IR sensor. The non-invasive pressure sensor is located in the swirling cover of the Monowave 300unit. With the simultaneous pressure and IR temperature feedback signals, microwave digestion program can be precisely controlled and monitored. Cooling of the reaction mixture is provided by leading compressed air along the vial through microwave cavity. The cooling process starts automatically after the heating program is completed.

To increase the sample throughput, a similar MWAD experiment of the pharmaceutical samples was performed with a MARS Xpress Microwave Digestion System (MARS 5, CEM Corporation, Mathews, NC, USA). The system was equipped with a rotor capable of holding a maximum of 12 high pressure PTFE sample digestion vessels (XP 1500) operable at temperatures of up to 260°C and 500 psi. Temperature of the microwave program was achieved through feedback via fiber optic sensor fitted with control vessel and a pressure transducer for controlling the microwave program. Sufficient time was given to allow pressurized vessels to come to room temperature before opening to atmosphere. To avoid cross-contamination, the PTFE vessels were thoroughly cleaned before use and between each batch of digestion experiments.

The quantitative determination of bismuth in pharmaceutical products, after digestion through the proposed MWAD procedure, was carried out using an Atomic Fluorescence Spectrometer (AF420, P.G. Instruments Limited, U.K.) equipped with an autosampler. The excitation source used in the instrument was a high intensity bismuth hollow cathode lamp operated with a primary current of 40mA and boosted current of 40mA. The instrument is controlled by the AF Win software. The argon gas source provides constant pressure environment to achieve constant liquid supply and the solenoid valves control sample timing sequence. The carrier liquid 2% (v/v) HCl takes sample (in sample loop) and mixes with NaBH₄ in the gas-liquid separator, where they react to produce the gaseous hydride. The argon gas sweeps the bismuth hydride from the gas liquid separator into the quartz tube atomizer and gets atomized when heated electrically by a tungsten filament to 200^oC, producing fluorescence at 223nm. The detector records the intensity of fluorescence signal. The data is acquired in time resolved mode using the AF Win data acquisition software. The area under the peak is used for the quantification of bismuth. The gas-liquid separator is provided with a magnetic stirring for efficient mixing of sample and reducer. This results in more complete reaction and also improves the efficiency of hydrides outflow to the atomizer. In addition the gas liquid separator is Peltier cooled, which minimizes the water content of the hydride, thereby greatly reducing the fluorescence signal scattering interference and effectively reducing the fluorescence quenching. The optimized HG-AFS operating conditions that yielded the best sensitivity for bismuth are given in Table 1.

Table 1: Operating parameters of the AFS instrument.

Parameter	Setting
Bi Hollow Cathode Lamp Wavelength	223 nm
Negative high voltage of photomultiplier	280 V
Lamp primary current	40 mA
Lamp boost current	40 mA
Flow rate of carrier gas (Ar)	600 mL min⁻¹
Flow rate of auxiliary gas (Ar)	300 mL min⁻¹
Atomizer temperature	200 ^◦C
Atomizer height	7 mm
Signal recording mode	Peak area
Signal Integration time	20 s

The residual carbon content (mg L⁻¹) in the final digested solution after the proposed MWAD procedure was determined by ICP-OES (Ultima II, Horiba Jobin-Yvon, France) equipped with a cross-flow nebulizer coupled to a cyclonic spray chamber, using the emission line 247.856nm.

Chemicals and Reagents:

All chemicals were of analytical reagent grade unless stated otherwise. Concentrated nitric acid 69% (Emsure^® ACS, Merck, India) and concentrated hydrochloric acid 37% (Emsure^® ACS, Merck, India) was purified by sub-boiling in quartz stills in a class 10 clean bench and used wherever necessary. A 30% H₂O₂ (Merck, India) was used during the MWAD process. Sodium tetrahydroborate solutions were prepared by dissolving the appropriate amount of NaBH₄ powder (Sigma Aldrich, USA) in 0.2% (v/v) NaOH (GR grade, Merck, India). This solution was prepared daily and filtered through a 0.45µm membrane from Phenomenex. Ultra-pure water with >18.2 MΩ.cm resistivity, obtained using a Milli-Q Element water system (Millipore, Bedford, MA, USA), located in a class 100 area, was used for dilution of standards, for preparing samples and reagent solutions and for final rinsing of the acid cleaned vessels. All sample preparations were carried out in a class 10 clean bench. An intermediate stock standard solution of bismuth (1mg L^-1in 5% (v/v) HNO₃) was prepared by appropriate dilution of 1000 mg L^-1 single-element stock standard solution of bismuth (Merck). A set of working standard solutions ranging from 0 (blank) – 20µg L^-1 for bismuth was prepared by appropriately diluting the intermediate stock standard in 2% (v/v) HCl and used for the generation of calibration curves followed by its quantification with HG-AFS. The residual carbon content in the sample digests was determined using carbon standard solutions (20 to 100mg L⁻¹) prepared by dissolution of citric acid (Merck, India) in water. The bismuth-containing pharmaceutical samples – Pylobis, Stomafit and Bismuthum Subnitricum (3X), in tablet form were purchased from a local pharmacy.

Sample Preparation:

10 tablets of each medicine were weighed to find the average weight. The tablets were then pulverized into a fine powder using a mortar and pestle. The powdered sample was stored in an air tight container until use.

Microwave-assisted digestion procedure:

An accurately weighed aliquot (~0.1g) of the powdered sample, weighed to the nearest 0.1mg, was transferred into a 30mL glass vial of microwave digestion unit equipped with an appropriate magnetic stir bar and added 10mL of a mixture of 5% (v/v) HNO₃ to it. Then the mixture was shaken gently to ensure that the entire sample was completely wetted and left for 5 min to allow for pre-digestion. The glass vial was sealed with a PEEK snap cap and a PTFE-coated silicone septum just with the click of a thumb. The Monowave 300 system was set at maximum installed power of 850 W with a pressure limit (~30 bar). Then the glass vial was placed inside the microwave cavity and its contents were then subjected to microwave irradiation using the microwave program given in Table 2. The temperature of the glass vial was initially ramped to 180 ⁰C in 3 min and held there for 10 min. At the end of the microwave program, a stream of air passes along the walls of the reaction vessel, allowing its rapid cooling. The vials are automatically depressurized with air circulation and cooled down to 60 ⁰C during the opening process of the cavity cover. The temperature of the liquid phase during microwave irradiation was monitored by using an infrared sensor located at the left side of the cavity. A clear solution with negligible residue was obtained at the end of microwave digestion process. The sample digest was appropriately diluted with 2% (v/v) HCl to obtain the concentration of bismuth within the calibration range and analyzed by HG-AFS.

Table 2: Microwave assisted digestion program.

Step	Program	Temperature (°C)	Time (min)	Cooling	Stirrer Speed (rpm)
1	Ramp	180	3	Off	600
2	Hold	180	10	Off	600
3	Cool down	60	0	On	600

Before starting each digestion experiment, microwave vessels were cleaned with 10% (v/v) HNO₃ to prevent cross-contamination from previous set of samples. With each series of digestions, corresponding process blanks without addition of sample, were also subjected to the same procedure and analyzed. Three aliquots of each sample were subjected to the MWAD procedure and the results were given as the average of at least 3 replicates.

A four point aqueous calibration was used in the quantification of Bi and standard addition method was also applied, in order to test possible matrix interferences, if any.

RESULTS AND DISCUSSION:

In the development of the MWAD procedure, experimental variables such as effect of oxidant composition, its concentration and microwave operation parameters (digestion time and temperature) were evaluated in order to obtain the maximum efficiency of digestion thereby obtaining quantitative recovery of bismuth in pharmaceutical products. The efficiency of each optimization step was evaluated by calculating the percentage recovery of bismuth, expressed as the ratio of the determined concentration values, relative to the value calculated as per the formula weight. An oxidant volume of 10mL was employed in all the microwave-related experiments. Final values of bismuth were calculated based on dry weight basis. The pharmaceutical medicine – Pylobis purchased from local pharmacy was used as a representative sample in all the optimization steps described below. According to the manufacturer, each Pylobis tablet contains colloidal bismuth (III) subcitrate equivalent to 120mg of Bi₂O₃ and other excipients. Each tablet of Pylobis weighs about 0.62g and the concentration of bismuth based on the formula weight was calculated to be 174mg/g.

Optimization of NaBH₄ and HCl concentration for BiH₃ formation:

The effect of NaBH₄ and HCl concentration on the fluorescence signal of bismuth by HG-AFS was evaluated. In all the optimization experiments the concentration of bismuth was kept constant at 10µg L^-1. The criteria for choosing the optimum conditions were based on the maximum signal-to-background ratio. However, in this study, the Bi blank values was very low and remained constant when different concentrations of NaBH₄ and HCl were used. Hence the hydride generation conditions were optimized using only signal intensity. The concentration of NaBH₄ was optimized between 0.5 – 1.5% (m/v) in 0.2% (m/v) NaOH, using 3% (v/v) HCl as the carrier solution. The fluorescence intensity remained constant when the NaBH₄ concentration was in the range of 1.1-1.5% (m/v), but the sensitivity decreased rapidly below 1.1%. Hence 1.2% (m/v) NaBH₄ was chosen as the optimum concentration for further experiments.

The effect of various HCl concentrations on the signal intensities was investigated in the range 1 – 4% (v/v), using the optimized NaBH₄ concentration of 1.2% (m/v). The analytical signal was maximum when 2% (v/v) HCl was used as the carrier solution and thereafter the signal decreased gradually. This could possibly be due to the generation of excess amount of hydrogen at higher HCl concentrations which dilutes the bismuth hydride concentration in the atomizer and therefore decreases the intensity of the Bi fluorescence. Therefore an HCl concentration of 2% (v/v) was chosen for the carrier and sample solution acidity.

Development of MWAD method:

The wide range of pharmaceutical drugs often contains complex organic compounds as the main active ingredient along with other excipients. In bismuth-containing pharmaceuticals, bismuth exists as organic or inorganic coordination complexes³. The knowledge of the composition of the pharmaceutical product is therefore important in selecting the appropriate reagents for complete mineralization of the sample. In addition the above solvent mixture should also be compatible with the instrument of choice (AFS/ICP-OES in this case).

Evaluation of different acids for sample digestion:

The wet digestion of a material with oxidant reagents either by conventional heating or by means of microwave radiation is the most common approach for complete mineralization of sample (pharmaceutical product in this case)^32,33. The acid or combination of acids is chosen for its efficiency in decomposing the matrix. The digestion mixture plays an important role in the destruction of the compounds in the matrix; but it must also ensure that the analytes are soluble and are retained in solution in a stable form for analysis³⁴. Among the oxidants, nitric acid is most commonly used because of its suitable oxidizing power, the possibility of obtaining it at high purity or of purifying it by sub-boiling distillation, and also because several elements will be in solution as soluble nitrates. Mixtures of HNO₃ with H₂O₂, HCl and HF, are also used, depending on the matrix, analytes, and/or digestion system³⁵.

Studies reported by various authors including our group^23-31,36 have shown that dilute acids absorb microwave radiation more efficiently than concentrated acids thereby achieving more quantitative recovery of a wide range of elements in various solid matrices. Additionally, the use of dilute acid solutions is one of the parameters associated with “Green Chemistry” principles^20-22. In the first set of experiments, to investigate the mineralization efficiency, the Pylobis pharmaceutical, used as a representative sample was digested using only dilute HNO₃ and also in combination with other reagents. Based on the results obtained from preliminary experiments, 10 mL each of the following reagent mixtures were tested for MWAD of the pharmaceutical sample – 5% (v/v) HNO₃, 5% (v/v) HNO₃ + 2.5% (v/v) HCl, 5% (v/v) HNO₃ + 2.5% (v/v) H₂O₂, 5% (v/v) HNO₃ + 2.5% (v/v) H₂O₂+ 2.5% (v/v) HCl. Each digestion was performed in triplicates using the microwave digestion program given in Table 2. The results obtained from the above experiments are given in Table 3. As seen here the % recovery of bismuth ranged from 86 to 99%, where different acid mixtures in dilute concentrations were used in the digestion. These results clearly indicate that dilute acids are sufficient to quantitatively recover bismuth from the pharmaceutical product using microwave irradiation.

Table 3: Concentration of bismuth obtained for Pylobis pharmaceutical using different acid oxidant mixture.

Acid Composition	Value obtained ± std. dev^a (mg g^-1)	% recovery^b
5% (v/v) HNO₃	172 ± 4	98.9
5% (v/v) HNO₃ + 2.5% (v/v) H₂O₂	168 ± 5	96.6
5% (v/v) HNO₃ + 2.5% (v/v) HCl	150 ± 6	86.2
5% (v/v) HNO₃ + 2.5% (v/v) H₂O₂+ 2.5% (v/v) HCl	169 ± 6	97.1

^an = 3. ^b Value given by the manufacturer = 174 mg g^-1.

In the above experiments the use of 10 mL of 5 % (v/v) HNO₃ alone was found to be sufficient to get quantitative recovery (99 %) for bismuth. To investigate whether similar recovery could be achieved by further lowering the concentration of nitric acid (i.e., < 5%), a set of experiments were carried out to optimise the concentration of nitric acid. The concentration of nitric acid was varied from 1 % to 5 % (v/v) in steps of 1.0 %, taking 10 mL of each solution to digest ~0.1 g of the sample. The % recovery of bismuth was found to be less (< 90 %) when the sample was digested with less than 5% (v/v) HNO₃, showing reduced oxidising potential. The concentration of nitric acid [< 5% (v/v)] was not sufficient enough to extract bismuth from the matrix into solution at 180 °C. The residue left behind was also higher post MWAD. Hence for all further experiments, 10 mL of 5% (v/v) HNO₃ was used for microwave digestion of the pharmaceutical samples containing bismuth.

The efficiency of dilute HNO₃ solutions in the oxidation of organic matter was well demonstrated and explained by Bizzi et al.^37,38 and Castro et al.³⁹. This was attributed to the regeneration of this acid promoted by the oxidation of NO to NO₂ and the absorption of this latter compound in the solution followed by its disproportioning reaction. The temperature gradient inside the microwave-assisted heated vessels during the initial steps of the sample digestion also played a fundamental role in the regeneration of nitric acid.

Determination of Residual Carbon Content:

For samples mainly containing organic constituents, the efficiency of the digestion may be established by determining the residual carbon content (RCC) in the final digest⁴⁰. The microwave digested solutions were analyzed for RCC by ICP-OES. The results are shown in Fig. 1. The RCC was highest in [5% (v/v) HNO₃+ 2.5% (v/v) HCl] mixture (1410 mg L^-1) which explains the comparatively lower % recovery for bismuth (Table 3), due to its lower oxidizing power. The lowest carbon content (688 mg L^-1) was found in [5% (v/v) HNO₃+ 2.5% (v/v) H₂O₂] mixture indicating its higher oxidizing capability. The RCC in the 5% (v/v) HNO₃ digests (1113 mg L^-1) was approximately 62% higher when compared to [5% (v/v) HNO₃+ 2.5% (v/v) H₂O₂] mixture, which was very obvious. But quantitative recovery of bismuth was obtained in both cases. In keeping with the green chemistry principles, the lesser the number of reagents used, the more greener the method. Hence 5% (v/v) HNO₃ solution was preferred as the digestion medium in the present study. The concentration of bismuth in the pharmaceutical products analyzed here are present at higher mg levels. Hence the sample digests were diluted sufficiently with 2% (v/v) HCl to parts per billion levels for the analysis of bismuth by HG-AFS. Due to higher dilution, no carbon related interferences was observed in the developed method. This was further confirmed by standard addition experiments. Nevertheless the information regarding the level of RCC in the final acid digests gives an idea about the suitability of the selected reagent for efficient recovery of analyte from the organic matrix by microwave irradiation and also look for any possible carbon related interferences during its determination.

Fig. 1: Residual carbon content (mg L^-1) in Pylobis sample digested with different oxidant mixtures

(sample weight = ~0.1 g, volume of oxidant = 10 mL, temperature = 180 °C, hold time = 10 min).

Optimization of microwave assisted digestion temperature and hold time:

The digestion temperature and time are the two most important microwave parameters for the quantitative recovery of elements from pharmaceutical products. It is well known that efficiency of sample digestion/decomposition to obtain quantitative recovery of various elements is closely related to the temperature reached by the solution during microwave irradiation. Similarly sample heating time should be as short as possible for complete decomposition of the matrix to increase analytical frequency. Hence experiments to study the effect of microwave temperatures and for different lengths of hold time were carried out while keeping the optimized composition of 10 mL of 5% (v/v) HNO₃ as oxidant and sample weight (~0.1 g) constant. At the end of each microwave digestion process, the sample digest was diluted to required volume with 2% (v/v) HCl for subsequent analysis of bismuth by HG-AFS.

Preliminary experiments using a microwave digestion temperature of 180°C with a hold time of 7 min, the recovery of bismuth was found to be close to 89%. In order to get maximum recovery of Bi, the maximum digestion temperature and hold time at the maximum temperature were optimized. We have carried out a factorial (two factors, three level) experimental design approach and determined the % recovery of bismuth after each digestion. The base level was chosen as 180 °C and 10 min. The upper and lower levels were obtained using a difference of ± 20 °C and ± 3 min from the base level. The results are shown in Fig. 2. The % recovery of bismuth was maximum (98%) at the base level of 180 °C for 10 min of hold time. At 200 °C, and > 7 min hold time, the pressure inside the microwave vessel reached the maximum set limit of 30 bar and the microwave program stopped before completing the set hold time. This explains the lower percentage recovery (< 91%) of bismuth at 200 °C compared to the recovery obtained at 180 °C. Hence a temperature of 180 °C and 10 min hold time was selected as the optimum and used in all the experiments.

Fig. 2: Percentage recovery of bismuth in Pylobis as a function of hold time and temperature

(sample weight = ~0.1 g, oxidant = 10 mL of 5% (v/v) HNO₃)

The RCC was analysed in all the above digests by ICP-AES and the results are shown in Fig. 3. The concentration of residual carbon was in the range from 600 to 2300mg L^-1. It is well known that higher the temperature and longer the microwave irradiation time, leads to better destruction of the organic matrix using an oxidant reagent. The same was observed here in the microwave digestion of bismuth-containing pharmaceutical products.

Fig. 3: Effect of temperature and hold time on the concentration of residual carbon

in the final digests of Pylobis (sample weight = ~0.1 g, oxidant = 10 mL of 5% (v/v) HNO₃)

Analytical Characteristics of the method:

The analytical figures of merit for bismuth were evaluated under the optimal experimental conditions of the developed MWAD method. Using the peak area as the quantitative parameter, a four point linear calibration plot for bismuth was obtained using standard solutions containing known amounts of bismuth in 2% (v/v) HCl, in the following concentration range: analytical blank, 2.0, 5.0, 10.0, and 20.0 µg L^-1 and was used for the quantification of bismuth in all the pharmaceutical samples after applying the developed MWAD procedure. The calibration curves in the above ranges exhibited good linearity with regression coefficients, R² > 0.999. Limits of detection (LOD = 3*S_b/m) and quantification (LOQ = 10*S_b/m) were determined as per IUPAC recommendations, using standard deviation of process blank solution measurements (S_b, n = 7) and the slope of the calibration curve (m). The LOD and LOQ were found to be 0.024 and 0.08 mg/kg respectively. The concentration of bismuth in the Pylobis pharmaceutical sample based on replicate determinations was found to be 172 ± 4 mg/g, which is in good agreement with the value calculated as per formula weight (174 mg/g). The reproducibility expressed as the relative standard deviation (RSD) values obtained from replicate analysis of the pharmaceutical sample after taken through the proposed MWAD procedure was 2.3 %.

Determination of bismuth by HG-AFS is well-known to be susceptible to interferences from various diverse elements, especially transition elements such as Cu, Ni, Co, which can also compete with the analyte for reaction with reducing agent⁴¹. In the pharmaceutical products analysed here, bismuth is the major element present, which is being determined. All other concomitant elements are generally known to be present in trace levels in pharmaceutical products^42,43. The digested sample solutions were diluted to parts per billion levels of bismuth; hence we do not expect the usual interference from any concomitant elements if present, during bismuth hydride generation. To test the matrix related interferences, if any, during the determination of bismuth by HG-AFS, standard addition method was carried out by spiking a known amount of bismuth to sample solution post MWAD process. In all the cases, quantitative recovery (> 98%) was obtained using aqueous calibration standards during its determination by HG-AFS. This implies that there were no significant interferences due to carbon matrix or concomitant elements present if any, which clearly demonstrates the efficacy of the proposed MWAD method. Therefore linear calibration using the aqueous standards was reliably applied for all quantifications without needing standard addition approach.

In the present MWAD method, with use of single vessel microwave Monoreactor system, the time needed for complete digestion process for each sample was about 15 min (including cooling time). With this setup, it is possible to analyse up to 2 samples in duplicate per hour and about 16 samples in 8 h. To increase the sample throughput, CEM MARS 5 microwave system was also used for digestion keeping all the experimental conditions [sample weight (~0.1 g), oxidant concentration [5% (v/v) HNO₃] and microwave program (180 °C/10 min)] constant. Quantitative recoveries in the range of 96-101% were obtained with CEM microwave system as in the case of single vessel microwave reactor indicating the efficacy of the method. However, simultaneous processing of multiple samples is here possible with CEM microwave unit which can hold up to 12 PTFE digestion vessels. In this case, the time needed for a set of 6 samples in duplicate was about 60 min (including microwave heating and cooling of vessels). With this setup, it is possible to digest about 40 to 45 samples (in duplicate) in an 8 h workday making the method very promising for the determination of bismuth in pharmaceutical samples.

Comparison of developed MWAD method with reported methods:

There are only a few reports on the determination of bismuth in pharmaceuticals in the literature. A comparison among the sample preparation procedures reported in these works and the present MWAD method is shown in Table 4. A majority of these methods have employed conventional digestion techniques; using a hot plate for sample decomposition, thereby requiring high amounts of concentrated acids or acid mixtures (5 – 25 mL) and long decomposition/dissolution times for complete processing of the sample material^9-18.

Table 4: Comparison of the proposed microwave-assisted digestion method with reported analytical procedures for the determination of bismuth in bismuth-containing pharmaceuticals.

Title of the reported procedure	Reagents used/volume [sample weight]	Digestion method (Time)	Determination method	Ref. No.
Spectrophotometric determination of bismuth in pharmaceutical samples by extraction of the tetraiodobismuthate (III) anion into propylene carbonate	5mL 20% H₂O₂, 10mL 70% HClO₄	Hot plate	Spectrophotometry	9
Flow-Injection Analysis Spectrophotometric Determination of Bismuth in Environmental and Pharmaceutical Samples	25mL aqua regia, 5mL concentrated HCl, 50mL H₂SO₄ (0.1 mol L^-1), [1.0g]	Hot plate	FIA-Spectrophotometry	10
Determination of bismuth in pharmaceutical products using methyltriphenylphosphonium bromide as a molecular probe by resonance light scattering technique	1: 1 HNO₃	Heating at 700 °C (6 h)	Resonance light scattering technique	11
Selective Ion Flotation Separation and Concentration of Ultra Trace Amounts of Bismuth Using Arsenazo III and Its Determination by Inductively Coupled Plasma-Atomic Emission Spectrometry	10mL concentrated HNO₃, 2mL HClO₄, [0.14 – 0.4g]	Hot plate	ICP-AES	12
An on-line preconcentration/separation system for the determination of bismuth in environmental samples by FAAS	5mL 65% (w/w) HNO₃, 2mL HClO₄ [0.01g]	Hot plate	FAAS	13
Determination of Trace Amounts of Bismuth in Pharmaceutical and Water by Adsorptive Cathodic Stripping Voltammetry in the Presence of Xylenol Orange	10mL 0.1 mol L^-1 HNO₃	Hot water bath	Adsorptive Cathodic Stripping Voltammetry	14
Multi-walled carbon nanotube modified with1-buthyl3-methyl imidazolium hexaflouro phosphate supported on sawdust as a selective adsorbent for solid phase extraction of Bi (III)	5mL concentrated HNO₃ [0.01g]	Hot plate	FAAS	15
Dispersive liquid–liquid microextraction for the microvolume spectrophotometric determination of bismuth in pharmaceutical and human serum samples	5mL concentrated HNO₃	Hot plate	DLLME-Spectrophotometry	16
Dispersive Liquid-Liquid Microextraction of Bismuth in Various Samples and Determination by Flame Atomic Absorption Spectrometry	10-25mL 65% (w/w) HNO3, 4-10mL 70% (w/w) HClO₄ [2.0 – 5.0g]	Hot plate	DLLME-FAAS	17
Determination of bismuth (III) in environmental and pharmaceutical samples using an organic reagent	25mL aqua regia, 5mL concentrated HCl, 50mL H₂SO₄ (0.1mol L^-1) [1.0 g]	Hot plate	Nanodrop- Spectrophotometry	18
Determination of bismuth in pharmaceutical products using liquid–liquid extraction in a flow injection system	10mL concentrated HNO₃, 2mL 5% H₂O₂, [0.2-0.5 g]	Microwave (30 min)	FIA-LLE-Spectrophotometry	44
Development of a Single-Step Microwave-Assisted Digestion Method using Dilute Nitric Acid for Determination of Bismuth in Bismuth-containing Pharmaceuticals by Hydride Generation-Atomic Fluorescence Spectrometry	10mL 5% (v/v) HNO₃, [0.1 g]	Microwave (15 min)	HG-AFS	Present Method

MWAD is based on desorption of analytes from the matrices by non-ionizing microwave energy (normally at 2.45 GHz) with high digestion rates due to fast heating, low volume of acid reagents and reduced sample contamination due to sample treatment in closed vessels¹⁹. As seen here, 10mL of dilute nitric acid [5% (v/v)] was found to be sufficient to recover bismuth quantitatively from 0.1g of pharmaceutical samples in 15 min. Hence high amounts of concentrated acids are not required in the present approach which resulted in lower laboratory residues and these features are in agreement with the “Green Chemistry” recommendations. The prominent advantages of the developed single-step MWAD method was the use of very dilute nitric acid, rapidity, low running costs, safety and simplicity.

Method validation and analysis of other Bi-containing pharmaceutical samples:

The developed MWAD method was applied to the determination of bismuth in two other pharmaceutical tablets of which one is an allopathic medicine (Stomafit) and other is homeopathic medicine (Bismuth subnitricum 3X). These medications are used for gastritis and non-ulcer dyspepsia associated with H. pylori. The average weight of a single tablet of Stomafit and Homeo medicine was about 1.13 and 0.25g respectively. Each tablet of Stomafit contains Bismuth Subsalicylate USP 270mg (C₇H₅BiO₄) and the homeopathic medicine contains bismuthum subnitricum 3X [Bi₅O(OH)₉(NO₃)₄]. Based on the formula weight, the concentration of Bi was calculated per gram of the sample, as given in Table 5. For homeopathic medicine the 3X dilution (1000 times) was taken into consideration while calculating the concentration of Bi.

Table 5: Determination of bismuth in pharmaceutical products (n = 3).

Drug Name	Present method (mg/g)	Value given by manufacturer (mg/g)	Value obtained by reported method^a (mg/g)
Pylobis (Bismuth Subcitrate)	172 ± 4	174	173 ± 3
Stomafit (Bismuth Subsalicylate)	140 ± 3	138	142 ± 2
Bismuthum Subnitricum 3X (Homeophathic)	0.70 ± 0.05	0.73	0.68 ± 0.04

^aRef No. 18

A portion (~0.1g) of the powdered tablet was accurately weighed and taken through the developed MWAD method and analysed for Bi by HG-AFS. The results obtained by the developed method are in reasonably good agreement with the value calculated based on the information given by the manufacturer, as given in Table 5. The relative standard deviation (RSD) values obtained from replicate analysis were 2% and 7% for Stomafit and Homeopathic medicine. The higher % RSD observed in the case of homeopathic sample may probably be due to lower concentration of Bi.

In the absence of a suitable reference material, the developed method was validated by processing the samples by an independent reported method¹⁸. Briefly, ~0.1g of each sample (in triplicates) was digested with 5 mL of aqua regia on a hot-plate and the excess HNO₃ was removed by heating with concentrated HCl (3mL) to almost dryness. The residue was dissolved in HNO₃ and made up to a known volume. The time taken for the above sample preparation was approximately 2 hours. An aliquot of the digest after appropriate dilution with 2 % (v/v) HCl was analysed by HG-AFS. The results are given in Table 5. Applying the Student’s t-test to the values obtained by the two methods, showed no significant differences at 95% confidence level.

Fig. 4 demonstrates the variation in microwave power, temperature and pressure vs. irradiation time of the Monowave 300 system with the data obtained during the MWAD process under optimized conditions, which emphasizes the trend in their variation. In closed vessel conditions, nitric acid can reach temperatures (180°C) much higher than its boiling point (120.5°C), by applying high microwave power. At this elevated temperature, substantial increase in oxidation potential is achieved and the reaction proceeds more rapidly⁶. The graph clearly shows that the pressure increases up to 16/ 25 bar when the temperature is increased from 35 to 180 °C in 3 min. This demonstrates that the organic matrix components present in these medicines decompose very rapidly during MWAD with an optimized oxidant concentration of 5% (v/v) HNO₃, due to its increased oxidizing power at elevated temperature. The pressure attained at the maximum temperature gives an indication of the amount of organic content in the three samples. At the maximum temperature of 180°C, the pressure reached for homeopathic medicine (25 bar) was found to be higher than the allopathic medicine (16 bar), indicating the presence of higher organic content in those samples.

Fig. 4: Comparison of the variation in the microwave digestion pressure of the

three pharmaceutical samples (sample weight = ~ 0.1g, temperature = 180°C,

hold time = 10min, oxidant = 10mL of 5% (v/v) HNO₃)

CONCLUSION:

The proposed single-step MWAD method using dilute nitric acid without the use of any additional auxiliary reagents, was found to be suitable for digesting bismuth-containing pharmaceutical products, followed by quantitative determination of bismuth by HG-AFS. 10 mL of 5% (v/v) nitric acid could efficiently digest the sample with low values of residual carbon content (1100 mg L^-1) and recovery of Bi > 98%. Considering some important parameters such as adherence to the principles of green chemistry, analyst safety and time, the proposed procedure has significant advantages when compared to the conventional hot-plate digestion methods employing large volumes of concentrated acids. The developed method is highly reproducible and can be utilized for the rapid determination of bismuth in pharmaceutical products on regular basis.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Sanjiv Kumar, Head, NCCCM for his constant support and encouragement.

CONFLICT OF INTEREST:

All authors have no conflicts of interest to declare.

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Received on 21.10.2020 Revised on 24.11.2020

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):87-97.

DOI: 10.52711/2231-5675.2021.00017