Application of nucleophilic substitution reaction for sensitive determination of heptaminol hydrochloride in pharmaceuticals

Abstract

A straightforward and sensitive spectrofluorimetric approach was established for the determination of heptaminol hydrochloride (HTM-HCl) based on the derivatization of the drug through its reaction with 5-dimethylaminonaphthalene-1-sulfonyl chloride (Dansyl chloride). The reagent underwent a nucleophilic substitution of its chlorine atom with HTM to give N-(5-dimethylaminonaphthalene-1-sulfonyl)-6-amino-2-methylheptan-2-ol. The highly luminescent derivative was extracted using methylene chloride and subjected to analysis at an excitation wavelength of 345 nm and an emission wavelength of 490 nm. The chemical reaction occurred within an aqueous environment buffered with a 0.1 M borate buffer solution adjusted to pH 10.5. Experimental findings indicate that the proposed method displays sensitivity and linearity across a concentration range from 0.03 to 2 µg mL^− 1. The method achieves lower detection and quantification limits of 0.016 and 0.048 µg mL^− 1, respectively. The analytical validation of this method followed the guidelines outlined by the International Council of Harmonization (ICH). This approach was applied effectively for quantifying the medication in both tablet and oral drops formulations available on the market, demonstrating excellent recovery of 98.95 ± 0.45 for tablets and 99.37 ± 0.24 for oral drops with no interference from excipients.

Graphical abstract

Introduction

Heptaminol hydrochloride is 6-amino-2-methylheptane-2-ol hydrochloride. It exerts a positive inotropic effect on the cardiovascular system resulting in increasing the coronary blood flow and slight peripheral vasoconstriction which makes the drug suitable for the management of orthostatic hypotension. The drug is also used for catecholamine weaning in septic shock [1,2,3,4].

Officially, HTM-HCl can be determined by a potentiometric titration method as mentioned in European pharmacopoeia [5]. On the other hand, various analytical techniques were reported for determination of the cited drug. Chromatographic methods such as HPLC with UV detection [6], HPLC fluorescence detection [7, 8], HPLC MS detection [9,10,11,12] and GC [13,14,15] often require thorough clean-up procedures. These processes can be labor-intensive and time-consuming, particularly when a mass spectrometer detector is utilized. Additionally, the instrumentation involved in these techniques is expensive, further contributing to the overall cost of the analysis process. The reported TLC [16] method comprised the initial step of extracting the sample with ether under alkaline conditions, followed by the subsequent reaction of the extracted HTM-HCl with 4-chloro-7-nitrobenzo-2,1,3-oxadiazole prior to the separation process.

The spectrophotometric techniques are always characterized by its simplicity [17]. However, spectrophotometric methods for analysis of HTM-HCl [18, 19] lack of sufficient sensitivity for determination of the studied drug. The reported conductometric method for analysis of HTM-HCl [20] is considered as non- selective technique as it could be affected by presence of contaminated ions as shown in supplementary Table 1. By looking to reported spectrofluorimetric methods for analysis of HTM-HCl [18, 21,22,23,24,25], these methods depend on using expensive reagents, elevated temperature or have a relative low sensitivity.

The main advantage of spectrofluorimetric techniques is its high sensitivity beside to the simplicity as cleared from many previous reported works [26]. The presence of amine function group in the structure of the drug allows the derivatization with dansyl chloride (DNS-Cl) yielding a highly fluorescent product [27,28,29,30,31,32]. This work presents a new spectrofluorimetric method for determination of the cited drug based on dansylation reaction. The proposed method is characterized by its simplicity, rapidity, sensitivity, cost-effectiveness, and compatibility with ambient temperature conditions. It is particularly well-suited for the analysis of the drug within tablet formulations and oral drops.

Experimental

Instrument

The spectrofluorimetric measurements were conducted using a Jasco FP-8350 spectrofluorimeter. The equipment was equipped with a 150 W Xenon-arc lamp and operated at a photomultiplier tube (PMT) voltage of 400 V. The emission and excitation monochromators were configured with a slit width of 5 nm, while a scan rate of 1000 nm.min^− 1 was selected for the measurement.

Materials and chemicals

DNS-Cl was procured from Sigma (St. Louis, USA). It was prepared by dissolving 20 mg in 100 mL of acetone giving a solution with a concentration of 0.02% w/v. Sodium bicarbonate, sodium hydroxide, boric acid, methylene chloride, methanol, isopropanol, ethyl alcohol, and 1-butanol were obtained from El Nasr Company for Chemicals (Cairo, Egypt). HTM-HCl was generously provided as a gift by PHARCO Company for Pharmaceuticals Industries (Cairo, Egypt). Corasore^® tablets containing 150 mg of the drug per tablet and Corasore oral drops with a concentration of 150 mg mL^− 1 of the drug were obtained from a local pharmacy in Egypt. Borate buffer preparation was carried out by mixing different amounts of NaOH and boric acid all in concentration of 0.1 M to obtain the target pH.

Standard drug solution

To get the stock solution of HTM-HCl, an exact quantity of 10.0 mg of the pure drug was carefully placed into a 100 mL volumetric flask. Subsequently, it was dissolved thoroughly in distilled water and mixed until completely uniform. The volume was then adjusted to the marked line using the same solvent. Following this step, portions of the prepared stock solution were diluted further to reach a final volume of 100 mL, achieving the desired concentration for subsequent use.

Procedure for construction of the calibration graph

Different concentrations of the drug solution ranging from 0.03 to 2.0 µg mL^− 1 were introduced into 10 mL glass tubes fitted with stoppers. Following this, 0.4 mL of borate buffer solution with a pH of 10.5 and 0.6 mL of the prepared DNS-CL solution were added to each tube. For thirty minutes, the tube was kept at room temperature. Following excitation at 345 nm, the fluorescent product was extracted using 3 mL methylene chloride for 3 times, and the fluorescence was measured at emission wavelength 490 nm. The test experiment was conducted beside a blank.

Procedure for tablets formulation

Twenty tablets of the commercial Corasore^® product were accurately weighed, ground, and rendered uniform. An amount corresponding to 10.0 mg of the medication was subsequently subjected to ultrasonic treatment with distilled water in a standard flask of 100 mL for duration of 20 min. Post-sonication, the solution underwent dilution to reach the mark, followed by filtration. The resulting filtrate was subsequently diluted to obtain solutions falling within the requisite concentration range, and the stipulated analytical methodologies were implemented.

Procedure for oral drops

An aliquot of oral solution was diluted with water to the required working drug solution. Different volumes of the resulting working solution covering the linear range were transferred to stoppered glass test tubes and general analytical procedures were applied.

Results and discussion

The presence of the primary amino group in the inherent aliphatic structure of HTM-HCl facilitates its derivatization with DNS-Cl, resulting in the generation of a fluorescent derivative, as illustrated in Fig. 1. Interaction with DNS-Cl initiates the conversion of the drug into a highly luminescent product. Following this reaction, the resultant fluorescent species is extracted into methylene chloride and its emission intensity is recorded at 490 nm after subsequent excitation at 345 nm with a stock shift of 145 nm (Fig. 2). It should be noted that, the excitation and emission wavelengths for both DNS-Cl and its product with HTM are relatively similar. However, their fluorescence intensities were completely different. The similarity in excitation and emission wavelengths arises from the structural integrity of the dansyl group, which retains its fluorophore characteristics in both forms. However, the fluorescence intensities differ significantly due to the removal of the chloride atom attached to the sulfonyl group in the reagent and its replacement with amino derivative. The presence of the chloride atom in the reagent greatly quench its fluorescence due to high electronegativity of the chloride that can withdraw electron density from the naphthalene moiety This can destabilize the excited state and reduce the fluorescence efficiency. In addition, chlorine is a heavy atom which can lead to increased non-radiative pathways for energy loss, such as vibrational relaxation, which decreases the amount of energy emitted as fluorescence. Furthermore, Chlorine atom can facilitate intersystem crossing to triplet states, where the molecule can lose energy through non-radiative processes instead of returning to the ground state via fluorescence.

Fig. 1

The suggested reaction pathway of HTM-HCl and DNS-Cl

Fig. 2

Fluorescence spectra of 2 µg mL^− 1 HTM-HCl – DNS-Cl reaction product and blank containing DNS-Cl (0.02% w/v)

Selection of optimum experimental parameters

A comprehensive investigation was conducted to assess the influence of various parameters such as pH, DNS-Cl volume, buffer volume, reaction duration, and choice of extracting solvent on the interaction between HTM-HCl and DNS-Cl. This inquiry aimed to identify the most suitable reaction conditions.

Influence of pH

The recommended procedure was performed using varied pH of the buffer solution in the range of 7.0–12.0 using 0.1 M borate buffer. The product fluorescence is directly proportional to pH elevation until reaching pH 9.5 and remains nearly constant till pH 11. The fluorescence intensity exhibits a notable decrease with a further rise in pH. So, pH of 10.5 was selected as the most suitable pH for this experiment (Fig. 3).

Fig. 3

Effect of pH of borate buffer on the observed RFI for the reaction product of 2 µg mL^− 1 HTM-HCl and DNS-Cl

Influence of buffer volume

To assess the impact of 0.1 M borate buffer volume on the fluorescence of reaction product, different aliquots ranging from 0.1 to 1.0 mL were examined. The findings revealed a rise in fluorescence intensity with increasing buffer volume up to 0.3 mL, after which the intensity plateaued until reaching 0.6 mL. Beyond this point, further volume increase led to a decline in fluorescence intensity. Consequently, 0.4 mL was identified as the most suitable volume for this experiment. (Fig. 4)

Fig. 4

Impact of borate buffer (pH 10.5) volume on the reaction of 2 µg mL^− 1 HTM-HCl with DNS-Cl

Influence of time

The fluorescence intensity of the resulting product was measured immediately and at different time intervals. (5–50 min.). Results showed that the reaction between the cited drug and dansyl chloride is highly dependent on time and were completed after 25 min. The product exhibited stability for an additional 15 min. Nonetheless, prolonged standing led to a significant inhibition of fluorescence, so 30 min was chosen as the most suitable time for the reaction. (Fig. 5).

Fig. 5

The impact of time reaction on the RFI of the product between 2 µg mL^− 1 HTM-HCl and DNS-Cl

Influence of DNS-Cl volume

A range of volumes of dansyl chloride, spanning from 0.1 mL to 1 mL, was scrutinized to evaluate their impact on the fluorescence intensity of the resultant product. Observations revealed a progressive enhancement in fluorescence as the volume of the reagent was incremented until reaching 0.5 mL. Subsequently, the fluorescence intensity remained relatively stable up to 0.8 mL. Nevertheless, a discernible decline in fluorescence intensity was noted upon the addition of higher volumes of the reagent. Consequently, 0.6 mL was identified as the most suitable volume for this experiment. (Fig. 6).

Fig. 6

The impact of varying volumes of DNS-Cl on the RFI of its reaction product with 2 µg mL^− 1 HTM-HCl

Influence of solvent

The fluorescence intensity of reagent blank in water was very high, therefore different solvent were tried to reduce the reagent blank reading. Thus, various water-miscible solvents were employed for dilution of the fluorescent product without extraction. The examined solvents were methyl alcohol, ethyl alcohol, isopropanol, and 1-butanol. However all these solvents also demonstrated elevated blank values and thus, were consequently eliminated from consideration. To address this issue, the product was subjected to extraction using methylene chloride which was chosen because of its higher extraction efficiency and its relatively lower toxicity compared to other organic solvents. In addition, the fluorescence intensity of the blank was relatively low as seen in (Fig. 2).

Validation of the suggested approach

The proposed method was validated following the ICH guiding rules [33] regarding the linearity, range, detection and quantitation limits, robustness, accuracy and precision.

Linearity and range

The provided analytical strategy was utilized to evaluate multiple drug solutions at different concentration levels. Through the plotting of relative fluorescence intensity at 490 nm against drug concentration in µg.mL^-1, a calibration graph was established for the spectrofluorimetric approach. Notably, the proposed method displayed excellent linearity within the concentration range of 0.03 to 2.0 µg.mL^-1. The data tabulated in Table 1 corroborate the high degree of linearity achieved with this approach.

Table 1 Analytical parameters for analysis of cited drug with the suggested method

The limit of detection (LOD) and limit of quantitation (LOQ) were determined following the guidelines provided by the International Council for Harmonization (ICH) [33] by applying the equation: LOD = 3.3σ/s and LOQ = 10σ/s whereas σ represent the standard deviation of intercept, while s is the slope of the graph. Value of LOQ was 0.048 µg mL^-1 while LOD was 0.016 µg mL^-1.

Accuracy

The accuracy of the suggested method was assessed by determining the % recovery of three distinct HTM-HCl concentrations. Each experiment was conducted in triplicate, and the results are presented as the mean percentage recovery along with the standard deviation in Table 2. The close alignment of the % recovery values with 100% across all investigated concentrations provides compelling evidence of the method’s accuracy.

Table 2 Accuracy evaluation for the proposed method

Precision

In order to assess both intraday and interday precision, three varying concentrations falling within the linearity range were tested using the proposed strategy. Intraday precision was evaluated by conducting experiments on the same day, whereas interday precision was determined by performing experiments over three successive days. All experiments were conducted in triplicate. The high precision of the proposed method was demonstrated by the low % RSD (relative standard deviation) values, as detailed in Table 3.

Table 3 Precision and accuracy evaluation of the suggested method at inter – and intra-day levels

Robustness

The method’s robustness was evaluated by implementing minor alterations to the experimental parameters including DNS-Cl volume, buffer volume, and pH, then computing the percent recovery. Data listed in Table 4 confirm that there was no significant effect for the small change of these parameters on method performance.

Table 4 Evaluation of method robustness

Application of the proposed method analysis of HTM-HCl pharmaceutical formulations

The spectrofluorimetric method investigated in this study demonstrated efficacy in estimating HTM-HCl in both tablet and oral drop formulations. Notably, high % recovery values of 98.9 ± 0.04 for tablets and 99.37 ± 0.24 for oral drops were attained. A comparative analysis between the proposed method and a previously reported approach [22] conducted through t-value and F-value calculations, indicated no significant deviation from theoretical values at a 95% confidence interval. This underscores the method’s adherence to accepted levels of accuracy and precision. Furthermore, the results presented in Table 5 corroborate the absence of interference from common excipients.

Table 5 Application to tablet dosage form and comparison with reported method

Comparison of the proposed method with the published fluorimetric methods

Six spectrofluorimetric methods [18, 21,22,23,24,25] were previously reported for the determination of HTM-HCl. A comparison of the current method with these published spectrofluorimetric methods is presented in supplementary Table 2. As shown in the table, four of these methods [18, 22, 23, 25] had lower sensitivity for HTM-HCl than the proposed spectrofluorimetric method. In addition, no heating step was need compared to the method based on condensation reactions [18, 22, 25]. Furthermore, the utilized reagent (DNS-Cl) is relatively cheap compared to the extremely expensive fluorescamine reagent [23]. Although the reported methods [21, 24] had a comparable analytical performance to the proposed method, the proposed method based on extraction of the fluorescent product to methylene chloride which has relative low toxicity compared to chloroform used in DAS derivatization method [21]. The proposed method also depends on using a single non expensive reagent compared to the orthophthalaldehyde/2ME method [24] with a larger stock shift. The excitation/emission wavelengths were 334/451 (Δλ = 117 nm) in the reported method and 345/490 nm in the proposed method. Thus, the proposed method has a very large Stock shift, (Δλ = 145 nm). Therefore, the proposed method has the merit of the, simplicity, and cost effective.

Conclusion

A highly sensitive spectrofluorimetric method was developed based on the dansylation of the primary amine of HTM-HCl with DNS-Cl. This method was deemed suitable for analyzing the drug in its pure form, tablet formulations, and oral drops. Notably, the approach is characterized by simplicity, rapidity, sensitivity, and the ability to be performed at ambient temperature without heating. The high sensitivity is evidenced by the low values of LOD (0.016 µg mL^− 1) and LOQ (0.048 µg.mL^− 1). As a result, it can be effectively applied for routine analysis of the drug in quality control units. The reported analytical techniques, the suggested method is superior on its sensitivity, simplicity and low cost. Although, chromatographic methods like HPLC (with UV, fluorescence, or mass spectrometry detection) and GC are effective, they require extensive clean-up, making them labor-intensive and costly. The reported TLC method was tedious, involving sample extraction with ether under alkaline conditions, followed by a reaction before separation. While spectrophotometric methods are simpler, they lack sufficient sensitivity. Additionally, a conductometric method is noted for being non-selective.