CFIA-ISNAG fluorimeter for the Determination of Bromhexine-HCl in Drugs via the Measurement Scattered Light at ± 90˚

A precise, simple, and accurate continuous flow injection technique was used for the instantaneous estimation of bromhexine hydrochloride (BH-HCl) in tablet dosage form. The chemical and physical parameters of the reaction of BH-HCl with tetraphenylborate to produce a yellowish-white precipitate were determined using an ISNAG fluorimeter analyzer and diverging light at 90°. The calibration curve for BH-HCl was linear with correlation coefficients of 0.9994 and linearity percentage r 2 % = 99.87 over a concentration range of 0.01-20 mmol/L, L.O.D = 0.3610 µg/125µL (0.007 mmol/L), and RSD% less than 0.3% for 3 and 13 mmol/L (five replicates). This approach was efficiently used to estimate the levels of BH-HCl in two distinct pharmaceutical companies. When the newly developed method was compared with the turbidimetric method using a t-test and standard addition, it was discovered that there had not been any significant difference between the two approaches at the 95% level of confidence.


Standard drugs and reagents
A standard solution of BH-HCl (0.1 mol/L) (SDI-Iraq) with the molecular formula C 14 H 20 Br 2 N 2 .HCl with a molecular weight of 412.6 g/mol was prepared by dissolving BH-HCl (4.126 g) in distilled water (100 mL).A stock solution of sodium tetraphenylborate (0.05 mol/L) (Fluka), with the molecular formula C 24 H 20 BNa, and a molecular weight of 342.22 g/mol, was prepared by dissolving sodium tetraphenylborate (8.5555 g) in distilled water (500 mL).

Sample preparation
Twenty tablets of BH-HCl (each tablet contains 8 mg of solvodine from Iraq and bisolven from Giza) were crushed and ground after being weighted (0.6042 g and 0.6370 g, equal to 0.04126 g of the active ingredient, 1 mmol/L), respectively.Each of the two types of samples was dissolved in distilled water.For removing any insoluble materials, the solution was filtered, the residue was rinsed with distilled water, and the volume was increased to 100 mL using the same solvent.

Apparatus
ISNAG fluorimeter analyzer: A homemade ISNAG (low-pressure mercury lamp) was used to measure the response, which is characterized by two lambdas (184.9 and 253.7 nm).A solar cell measuring 2[4× 2.5cm] was employed as the detector.
The flow system: Figure 2 shows the flow system to determine bromhexine-HCl.It consists of a peristaltic pump with two channels and variable speed (Ismatec, Switzerland).

Methodology
A flow injection manifold was employed, along with a two-line homemade ISNAG fluorimeter (Figure 2).The first line represents a carrier stream (HCl, 30 mmol.L -1 , 1.5 mL/min.)that introduces the sample segment BH-HCl (10 mmol.L -1 , 125 µL) into the reaction stream, which is combined with the second line (TPB, 0.04 mmol.L -1 , 1.5 mL/min.) to form a yellowish-white precipitate as an ion pair complex at the Y-junction, with an outlet for the reactant's ion pair product.The precipitate was determined using a low-pressure mercury lamp (ISNAG-fluorimeter), which gives 253.7 and 184.9 nm, respectively.Because of their high frequency, the two lines readily diverge.The divergence of such an incident light beam will be detected at 90 o using a 2.0 mm path length flow cell that spans 100 mm and a 2 [4 × 2.5 cm] solar cell.Scheme 1 depicts a proposed mechanism for the BH-HCltetraphenylborate reaction [29].

. The Influence of varying tetraphenyl borate concentration
At a flow rate of 1.3 mL/min (for both the reagent and carrier stream lines), a series of tetraphenyl borate (0.005-0.1 mmol/L) is utilized as a precipitating agent, BH-HCl (10 mmol/L), sample volume (85 µL), and open valve mode (time in seconds to load the manifold with the sample of the analyte).It can be shown that increasing the concentration of the reagent up to 0.04 mmol/L improves sensitivity, which is expressed as the peak response (Figure 3A), resulting in more precipitate particulates being formed, which might increase divergent light towards the solar cell.Above 0.04 mmol L -1 , the diverged light intensity decreased, which could be due to irregular agglomerate formation of precipitate particulate that prevented the diverged light from reaching the solar cell at 0-90˚ or a massive blockage that blocked the passage and reflected most of the incident light that was not detected by the solar cell.Table 1 shows all of the obtained results.Figure 3 shows that the obtained concentration of TPB is 0.04 mmol/L.On such a basis, HCl (50 mmol/L) was utilized as a carrier stream.

Effect of acidic media
Various acid solutions (50 mmol/L) for each of HCl, HNO 3 , H 3 PO 4 , H 2 SO 4 , and CH 3 COOH, along with H 2 O, were prepared to determine the suitable medium employed in the precipitation of BH-HCl (10 mmol/L) by TPB (0.04 mmol/L), which was used as the carrier stream in Figure 4.Because of the compactness of precipitated particles, an increase in the response sensitivity of the HCl medium as the carrier stream results in an increase in the divergence of light at 90 degrees.As a result, HCl was chosen as the best medium for this experiment, and all results are displayed in Table 2.  Figure 5B depicts an increase in diverged light with increasing HCl concentrations ranging from 5 to 30 mmol/L.It was noticed from Table 3 above 30 mmol/L that there was a decrease in response, which might be attributed to the dissociation of some precipitate particles.Therefore, 30mmol/L has been selected as the optimal concentration as a medium for precipitate formation.Figure 5A shows the response profile for variation in hydrochloric acid.

Physical variation 3.2.1. Flow rate
The BH-HCl (10 mmol/L)-TPB (0.04 mmol/L) system was used, and the sample volume was 85 µL.Different flow rates (0.7-2.0 mL/min) for the carrier stream and precipitating agent, respectively.Figure 6 shows that at the low flow rates, peak height increased up to 1.5mL/min, respectively, for the carrier stream and reagent, with wider peak widths, possibly because of dispersion and dilution, followed by a constant and subsequently a drop in peak height at flow rates higher than 1.5 mL/min.To get a regular response, narrow (∆tB), and limit reaction solution consumption, the optimal flow rate for a complete reaction between BH-HCl and TPB was 1.5 mL/min.Table 4 shows all of the obtained results.

Effect of different sample volumes
The BH-HCl (10 mmol/L)-[TPB] (0.04 mmol/L) system was used.Flow rate of 1.5 mL/min for carrier stream (HCl) and reagent in open valve mode.The injection volume was varied from 75-200 µL. Figure 7A and Table 5 show any increases in a sample volume of as high as 125 µL increased in the height of the responses, followed by a modest rise or a nearly constant response.This could be due to the elongation of the period leading up to the detection point because of the larger sample volume, along with the size or weight of the produced precipitate particles, which could result in a slight delay in the weight.As a result, 125 µl was the ideal sample volume.

Calibration graph
A series of BH-HCl concentrations that range from 0.01 to 20 mmol/L were prepared under the stated optimum conditions for preparing a scatter plot diagram, which is followed by the selection of a calibration graph; Figure 8A displays the response profile for the present investigation.The variation of scattered divergent light utilizing an ISNAG fluorimeter with a BH-HCl concentration is shown in Figure 8B.The obtained results for the variation of measured responses with BH-HCl concentrations show a linear range between 0.01 and 20 mmol.L -1 with a coefficient of correlation, r = 0.9994, indicating that an increase in the BH-HCl concentration results in increasing the precipitate with a smooth surface, which acts as a reflecting mirror to the detector, which will be measured at 90 degrees according to instrument design.This approach was compared to the traditional method of measuring turbidity using turbidimeter equipment (HANA).Table 6 shows the obtained results.

Repeatability and detection limit
Repeatability has been researched for determining BH-HCl through measurement of diverged light at 90˚ for the reaction of BH-HCl with TPB acid at the concentrations of 3 and 13 mmol.L -1 , as shown in Figure 9.The limit of BH-HCl detection is calculated by two methods.Gradual dilution of low concentrations in the graph of the calibration curve or based upon the slope's numerical value.Table 7 shows the obtained results.

Application of a developed method for the determination of BH-HCl in pharmaceutical preparations
Continuous flow injection analysis using a homemade ISNAG fluorimeter has been utilized for the determination of BH-HCl in two different pharmaceutical preparations (solvodine and bisolvine).A set of solutions has been prepared for every one of the pharmaceutical drugs (1 mmol/L) through the transfer of 1 mL to each of the five volumetric flasks (10 mL).This was followed by the addition of gradual standard BH-HCl volumes (0, 0.01, 0.02, 0.03, and 0.04 mL) of 0.1 mol/L for obtaining 0, 0.1, 0.2, 0.3, and 0.4 mmol/L when using the ISNAG fluorimeter and turbidimeter (classical method).The measurements have been carried out using both approaches.Figure 10 exhibits the profile of the response for the present work and standard addition graphs of calibration with the use of the ISNAGfluorimeter.The results have been solved mathematically with the standard addition approach.The results are listed in Table 8A, at a 95% confidence level, which shows practically concentration of BH-HCl in every one of the pharmaceutical drugs with the use of two different analysis approaches.Table 8B shows the practical content of the active ingredient at a 95% confidence level and the efficiency of determination, in addition to the paired t-test, which compares two different paths [30,31].
First: Individual t-test: Calculating the t-value to compare a newly developed approach (the ISNAG fluorimeter) to the quoted value (8 mg) listed in Table 8B column 6.It has been noted that the calculated t-value is less than the critical tabulated t-value in the case of using the ISNAG fluorimeter (developed method).This is an indication of the fact that there has not been a considerable difference between the quoted values of every one of the individual companies with t cal at 95% interval of confidence.Second: The paired t-test has been utilized for comparing the developed approach with using the ISNAG fluorimeter CFIA with the classical approach.The result that has been obtained indicates that clearly there has not been any considerable difference between the two methods, since the calculated t-value (-0.693) is less than t tab (4.303) for determining BH-HCl in the pharmaceutical preparations at 95% level of confidence, as can be seen from Table 8B.Xd: Difference between two methods,σ n-1 :Difference standard deviation, d: difference average value,µ: quoted value,n= no. of the samples = 2, t 0.025, = 1.96 at 95 %.

Conclusion
A new homemade instrument, ISNAG (low-pressure mercury lamp: 184.9 and 253.7 nm), was used for the estimation of bromhexine hydrochloride.The method works by measuring the precipitate formed by bromhexine hydrochloride and sodium tetraphenylborate when the drugs are pure and tablet dosage forms.When the newly developed method was compared to the traditional method (turbidimetric technique) using the t-test and standard addition method, it was discovered that there had not been any significant difference between the two approaches at the 95% level of confidence.

Figure 1 :
Figure 1: The structure of BH-HCl

Valve 6 :
medium pressure injection valve with sample loop (IDEX Co., US) (1 mm i.d.Teflon, variable length); as a detector, two [4 × 2.5 cm] solar cells were used to collect signals via sample travel over a 100 mm line of 2 mm optical aperture.Potentiometric recorder: Recorded the output signals (Siemens, Germany) (1V-5V, 1000mV-5000mV).Turbidimeter: The turbidity readings under batch conditions have been manufactured by the Hanna Company (US).

Figure 2 :
Figure 2: A flow injection diagram system of instrument analysis for determining BH-HCl

Scheme 1 :
Scheme 1 : A probable proposed mechanism for the BH-HCl reaction with tetraphenylborate

Figure 3 :
Figure 3: A. Response profile of bromhexine-HCl with various tetraphenyl borate concentrations B. Average peak heights vs. concentration of tetraphenylborate (TPB)

Figure 4 :
Figure 4: A. Effects of different acids that are utilized as carriers on the response profile

Figure 5 :
Figure 5: A. Effects of the hydrochloric acid that is utilized as acarrier stream on the response profile B. Average peak heights vs. concentration of hydrochloric acid

Figure 6 :
Figure 6: A. Diverged light versus time profile with the use of the variable flow rate B. Plot average peak heights vs. flow rate

Figure 7 :
Figure 7: Effects of sample volume upon: A. Response profile B. Output of the diverged beam of incident light using the BH-HCl-TPB system

Figure 8 -
Figure 8 -Calibration graph for variations of the BH-HCl concentration on: A. Response profile versus time B. Diverged light expressed by linear equation with the use of the ISNAG-fluorimeter.Residual (ȳi-Ŷi),ȳi: practical value, Ŷi: estimate value

Table 7 :
Repeatability and detection limit for BH-HCl at optimum parameters via divergence of light measured at 90˚ expressed in mV.value based upon the slope, S B = standard deviation of blank repeated for 13

Table 1 :
Effects of the concentration of the TPB upon the function of the response, which is represented as the average peak height

Table 2 :
Effects of various acids as carrier stream on the response function that is expressed as an average peak height

Table 3 :
Effect of HCl concentration on the response function that is represented as an average peak height

Table 4 :
Effects of flow rate on response function that is represented as an average height of the peak

Table 5 :
Effects of sample volume upon the response function expressed as an average peak height

Table 6 :
Summary of results for determining bromhexine hydrochloride using an ISNAGfluorimeter

Table 8A :
The results of standard addition for determining BH-HCl in two pharmaceutical preparations

Table 8B :
Summary of the results for the paired t-test, efficiency, and practical content for determining BH-HCl in two pharmaceutical preparation samples