Simple Potentiometric Strategy for the Detection of Levofloxacin Hydrochloride and Daclatasvir Dihydrochloride in Pure form and Pharmaceutical Preparations

Introduction

The use of electrochemical sensors has explored as a new class of chemical read-outs for monitoring a variety of chemical species because of their unique physico- chemical features and sensing properties.  Promising applications have been found by potentiometric approach in various pharmaceutical analysis ^1-3, diverse chemicals of food quality ⁴, clinical and biological interest ^5,6. Various analytical benefits of potentiometric sensors, including their high stability and electrical conductivity have been reported ⁷.

Levofloxacin hydrochloride (LVX) is a member of the family of medications known as broad spectrum antibiotics. It is commonly recommended in urinary tract infections, gastrointestinal tract.  Also, it is prescribed to treat the respiratory tract infection and pelvic inflammatory diseases ⁸. LVX (Figure 1a) is a member of quinolones, and its literature survey addressed some analytical techniques for the determination of LVX in different matrices. Among these methods are spectroscopic methods such as spectrophotometry ^{9- 15}. Furthermore, LVX was detected using high performance liquid chromatography ^16-19.

Daclatasvir dihydrochloride (DAC) (Figure 1b), is recommended for the treatment of hepatitis C virus by inhibiting NS5A protein. It is a new oral antiviral medication which exhibits a potential pangenotypic activity ^{20, 21}. Few chromatographic methods were reported, including high performance liquid chromatography ^22-28. The investigated drug was detected using spectrophotometric method ^29,30_. A chitosan modified electrochemical electrode for the detection of DAC was also, reported ³¹.

Figure 1: Chemical structures of (a): Levofloxacin hydrochloride and (b): Daclatasvir dihydrochloride Click here to View figure

 

The present study aimed to suggest two simple and accurate TBP-LVX and TPB-DAC plastic membrane sensors for LVX and DAC determination in their bulk powder and commercial formulations.

Experimental

Chemicals and Reagents

Pure grade LVX and DAC were provided by Pharmaceutical Co. (Memphise).  Daclenza^® (60 mg/tablet) and Unibiotic^® (500 mg/tablet) were purchased drug local drug stores.  Sodium tetraphenyl borate (TPB) purity of (99.5 %), dioctylphthalate (DOP, 98.0 %), Tetrahydrofuran (THF), hydrochloric acid 36%, methanol, Polyvinyl chloride (PVC), microcrystalline cellulose, L-histidine, L-cysteine, titanium dioxide, talc, starch, mannitol, lactose and magnesium stearate, were acquired from (Sigma-Aldrich, Hamburg, Germany). Sodium hydroxide, potassium chloride, sodium chloride, calcium carbonate, barium chloride and copper sulfate were supplied by (BDH laboratory supplies, Poole,UK).

Instrumentation

The potentiometric detections were performed under continuous magnetic stirring at 25±1^°C with a Jenway pH-meter. A saturated standard electrode; silver/silver Cl^– double junction electrode was used. The same model of pH meter was used for pH adjustment.

Preparation of Analytical Solutions

Stock LVX and DAC                                

A solution of each LVX and DAC was daily prepared by dissolving 1.98 g and 4.05 g in 50 mL water forming a concentration of 0.1 mol L^-1. Modelling analytes in the range of 1.0 10^-7-1.0 10^-1 mol L^-1 was daily obtained.

LVX and DAC Tablet Solutions

Not less than 10 tablets of each Daclenza^® (60 mg/tablet) and Unibiotic^® (500 mg/tablet) were finely agitated and 0.01 mol L^-1 tablet solution was formed from each LVX and DAC into 10 mL methanol. A clear solution was taken and completed to volume using distilled water, after complete centrifugation and filtration. Two ranges of working solution 1.0χ10^-5– 1.0χ10^-2 and 1.0χ10^-6-1.0χ10^-3 mol L^-1 were produced by dilutions using ultra pure water for LVX and DAC detection, respectively.

Preparation of LVX-TPB and DAC-TPB ion Pairs

The electroactive materials LVX-TPB and DAC-TPB of the developed sensors were prepared by the incorporation of equal volume of 0.01 mol L^-1 of each LVX, DAC and TPB was mixed. Resulted precipitates were filtered and dried at room temperature overnight.

Sensor Construction

Preparation of LVX-TPB and DAC-TPB Plastic Membrane Sensors

The conventional PVC membrane sensors of LVX and DAC were fabricated by adding 190 mg PVC to 10.0 mg of each LVX-TPB or DAC-TPB ion pairs and 0.35 mL of DOP as plasticizer. Approximately, 5.0 mL of tetrahydrofuran (THF) was used. Membrane contents were poured into rounded glass dish and left aside for drying. The semi-transparent PVC membrane was obtained.  The membrane is fitted with a polyethylene tube and the internal solution (1:1) 0.001mol L^-1 sodium chloride solution and LVX or DAC solution was used. Then, they were preconditioned in 0.001 mol L^-1 LVX or DAC solution for 1 hr ^32-34.

Sensor Calibration

Calibration graphs of the developed LVX-TPB and DAC-TPB sensors were plotted using modelling solutions of 1.0χ10^-7-1.0 10^-1 mol L^-1 LVX or DAC.

Standard Addition Method

The determination of each LVX and DAC in their commercial products was carried out using the standard addition method. It was conducted by dropping small additions to the analyte solution vs. the potential reading. The concentration of the test solution was obtained from(ΔE = E₂-E₁).

Results and Discussion

LVX and DAC sensors were fabricated using LVX-TPB or DAC-TPB electroactive materials. The sensitivity and selectivity of the constructed sensors were studied (Figure 2).

Figure 2: Typical calibration graphs of LVX-TPB and DAC-TPB plastic membrane sensors Click here to View figure

 

It was found that LVX-TPB and DAC-TPB sensors were displayed Nernstian responses (58.8±0.3 and 28.8±0.6 mV decade^-1) covering 1.0 10^-5-1.0χ10^-2 and 1.0χ10^-6-1.0χ10^-3 mol L^-1. The limits of detection 3.2χ10^-6 and 1.1χ10^-7 mol L^-1 were recorded for LVX and DAC sensors. The quantification limits were also evaluated and was found to be 9.7χ10^-6 and 3.4χ10^-7 mol L^-1 (Table 1).

Table 1: Critical analytical data of LVX-TPB and DAC-TPB PVCsensors

	LVX-TPB	DAC-TPB
Slope (mV decade-1)	58.8±0.3	28.8±0.6
Correlation coefficient, r	0.9989	0.9995
Interccept	434.03	305.88
Linearity range (mol L-1)	1.0χ10-5-1.0χ10-2	1.0χ10-6-1.0χ10-3
LOD	3.2χ10-6	1.1χ10-7
LOQ	9.7χ10-6	3.4χ10-7
Response time/s	30	40
pH	3.5-5.5	4.5-7
Life time/day	40	35
Temperature˚C	25˚C	25˚C
Accuracy (%)	99.7±0.7	99.2±0.4
Robbustness	99.2±0.4	99.7±0.3
Raggedness	98.6±0.7	99.4±0.1

 

The influence of three different kinds of plasticizers was carefully studied using DOS (ɛ = 4.0), DBS (ɛ = 4.5) and DOP (ɛ = 5.1).  DOP was superior to other plasticizers in terms of sensor performance owing to higher (ɛ = 5.1) of DOP (Table 2).

Table 2: The resulted slopes of LVX-TPB and DAC-TBP PVS sensors using different plastecizers

	LVX-TPB	DAC-TPB
DOS	49.9	18.5
DBS	50.4	23.8
DOP	58.8*	28.8*

 

*The optimum slope of fabricated sensors

The investigated solutions in the range of 1.0χ10^-7-1.0χ10^-1 mol L^-1 was used to calculate the response time. Response times for LVX-TPB and DAC-TPB were 30 & 40 s for lifetime 40 & 35 days (Figure 3).

Figure 3: Response time plot for (a) LVX-TPB and (b) DAC-TPB membrane sensors Click here to View figure

 

pH values for LVX-TPB and DAC-TPB sensors were evaluated using 1.0χ10^-3, 1.0χ10^-4 mol L^-1 and 1.0χ10^-4, 1.0χ10^-5 mol L^-1 solutions. The pH of the analyte solution was adjusted using dil. HCl. Then, dil. NaOH was added to gradually increase pH. The potentials were derived against pH. The constructed LVX-TPB and DAC-TPB were safely active in pH 3.5-5.5 and 4.5-7 (Figure 4).  The decrease in the pH below 3.5 and above 7 was attributed to the effect of H⁺ or OH^–, respectively.

Figure 4: pH influence on LVX-TPB and DAC-TPB sensors Click here to View figure

 

For investigating LVX-TPB and DAC-TPB selectivity coefficients, prepared sensors were employed to measure 1.0χ10^-3 mol L^-1 of each drug in presence of different possible interfering species using SSM³⁵.  Fabricated sensors selectivity coefficients were calculated using the following equation.

Log K^pot_drug  J ^z+= (E₂ – E₁) / S

No interference was noticed during the detection of LVX and DAC (Table 3).

Table 3:  Selectivity coefficients (K^Pot_Drug⁺) of LVX-TPB and DAC-TPB sensors

	KPotPLZ+
Interferent	LVX-TPB plastic	DAC-TPB plastic
	membrane sensor	membrane sensor
Na+	7.4χ10-3	1.1χ10-3
K+	6.8χ10-3	7.6χ10-4
Ca2+	1.5χ10-4	2.6χ10-4
Ba2+	2.3χ10-4	5.3χ10-4
Cu2+	3.5χ10-4	3.9χ10-4
Lactose	7.1χ10-3	1.2χ10-3
Talc	7.1χ10-3	1.0χ10-3
Starch	9.0χ10-3	2.8χ10-3
Mannitol	6.3χ10-3	3.9χ10-3
L. Histidine	2.5χ10-3	1.7χ10-4
L. cycteine	6.6χ10-3	2.7χ10-4
Magnesium stearate	5.6χ10-3	2.3χ10-3
Titanium dioxide	5.3χ10-4	8.9 χ 10-5
Microcrystalline cellulose	3.6χ10-3	4.2χ10-4

 

Technique Validation

It was conducted by evaluating various parameters in accordance with ICH guidelines ³⁶.

The linear relationship of the suggested potentiometric method was evaluated using LVX and DAC test solutions of concentrations 1.0χ10^-7  to 1.0χ10^-1 mol L^-1. The two fabricated LVX-TPB and DAC-TPB sensors were employed for the detection of LVX and DAC test solutions. The results obtained revealed a concentration linearity of 1.0χ10^-5-1.0χ10^-2, 1.0χ10^-6-1.0χ10^-3 mol L^-1 for LVX and DAC sensors, respectively.

Detection limits were evaluated when the slope was dropped by 17.9 mV. The results were 3.2χ10^-6 & 1.1χ10^-7 mol L^-1, while, quantification limits were 9.7χ10^-6 & 3.4χ10^-7 mol L^-1 for LVX-TPB and DAC-TPB, respectively.

Accuracy was examined in presenceof magnesium stearate using the standard addition method. The % recoveries were 99.7±0.7 & 99.2±0.4, for LVX-TPB and DAC-TPB sensors.

Precision was evaluated using intra-day and inter-day assay. RSD was less than 1% indicating good precision.

Table 4: Data obtained by investigating the precision of the fabricated LVX-TPB and DAC-TPB sensors

Intra-day assay				Inter-day assay
LVX-TPB		DAC-TPB		LVX-TPB		DAC-TPB
Taken*	% Recovery	Taken	% Recovery	Taken	% Recovery	Taken	% Recovery
5	99.8±0.4	5	99.3±0.2	5	99.6±0.3	5	99.0±0.3
4	99.0±0.6	4	99.6±0.1	4	99.4±0.5	4	98.4±0.8
3.3	98.8±0.8	3.3	99.7±0.3	3.3	99.2±0.2	3.3	98.9±0.6

 

*- log concentration, mol L^-1

Robustness was tested by minor changes in pH at 4.7±1.  Resulted data were 99.2±0.4 & 99.7±0.3 for LVX-TPB and DAC-TPB. Ruggedness was examined using (HANNA 211 pH meter). The obtained data were 98.6±0.7 & 99.4±0.1 for LVX and DAC.

Analytical Applications

Quantification of LVX and DAC

LVX and DAC were quantified directly in bulk form using LVX-TPB or DAC-TPB sensors. The listed data were 99.3±0.4 and 99.5±0.5 for tested sensors (Table 5).

Furthermore, LVX and DAC were estimated in their tablets; they were recovered by 98.9±0.3% and 99.3±0.6% (Table 6).

Table 5: Data obtained by the detection of LVX and DAC in bulk drug using LVX-TPB and DAC-TPB sensors

Sample	LVX-TPB PVC sensor			DAC-TPB PVC sensor
	Taken	Found	%	Taken	Found	%
Pure drug	–		Recovery			Recovery
	5	4.99	99.8	6	5.97	99.5
	4.3	4.28	99.5	5	4.98	99.6
	4	3.97	99.3	4.3	4.24	98.6
	3.3	3.26	98.8	4	3.99	99.8
	3	2.98	99.3	3.3	3.29	99.7
	2	1.98	99	3	3	100
%Mean ±SD	99.3±0.4			99.5±0.5
n	6			6
Variance	0.16			0.25
%SE	0.16			0.2
% RSD	0.4			0.5

 

Table 6: Data obtained by the detection of LVX and DAC in using LVX-TPB and DAC-TPB sensors using standard addition method

Sample	LVX-TPB PVC sensor				DAC-TPB PVC sensor
	Taken	Found	% Recovery	Sample	Taken	Found	% Recovery
Unibiotic® 500 mg /tablets	5	4.97	99.4	Daclenza® 60 mg/ tablet	6	5.95	99.2
	4.3	4.26	99.1		5	4.93	98.6
	4	3.95	98.8		4.3	4.28	99.5
	3.3	3.3	100		4	3.99	99.7
	3	2.99	99.7		3.3	3.28	99.4
	2	1.97	98.5		3	3	100
% Mean ±SD	99.3±0.6				99.4±0.5
n	6				6
Variance	0.36				0.25
% SE	0.24				0.2
% RSD	0.6				0.5

 

To evaluate the proposed method, the obtained results were statistically assessed using t- & F- tests at 95% confedence level ³⁷. The obtained results for LVX detection using the fabricated LVX-TPB was compared with Maleque el al. (2012) method in which LVX was detected using water: methanol: acetonitrile (9.0:5.0:0.5 v/v/v) as solvent at 292 nm ¹¹. Furthermore, the obtained results of DAC determination were compared with a simple spectrophotometric method which carried out by detecting the DAC drug in its bulk and pharmaceuticals, the detections were measured at 317 nm ³⁰. The outcome results revealed an excellent agreement with the previously mentioned published methods (Table 7).

Table 7: Data obtained for the detection of LVX and DAC in their pharmaceutical formulations by the proposed and reported methods

	Taken  mol L-1	Mean%±SD	n	Variance	%SE	%RSD	t-test	F-test
LVX-TPB	1.0 χ 10-5-1.0χ10-2	99.3±0.6	6	0.36	0.24	0.6	0.830 (2.228)*	1.78 (5.05)*
DAC-TPB	1.0χ10-6-1.0χ10-3	99.4±0.5	6	0.25	0.20	0.5	0.581 (2.228)*	1.96 (5.05)*
Reported     method of LVX 11	1-12 µg mL-1	99.7±0.8	6	0.64	0.32	0.8
Reported method of DAC 30	2-12 µg mL-1	99.6±0.7	6	0.49	0.28	0.7

 

* T_tabulted and F_tabulted³⁷

Conclusion

This present study described a selective potentiometry approach for estimation of LVX and DAC in their bulk and pharmaceutical forms. Sensors were accurate and precise for the assay of LVX and DAC and displayed excellent detection of the investigated drugs with lower detection limits.

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