Simple and Sensitive Colorimetric Method for the Determination of ...

DOI 10.1007/s11094-016-1449-3 Pharmaceutical Chemistry Journal, Vol. 50, No. 5, August, 2016 (Russian Original Vol. 50, No. 5, May, 2016)

SIMPLE AND SENSITIVE COLORIMETRIC METHOD FOR THE DETERMINATION OF MILNACIPRAN IN BULK AND SWELLABLE MATRIX TABLETS Talib Hussain,1,2 Muhammad Khurram Shahzad,1 Khezar Hayat,2 Khalid Hussain,1 and Nadeem Irfan Bukhari1* Original article submitted February 19, 2016. Milnacipran, a serotonin and adrenaline reuptake inhibitor used to treat fibromyalgia and major depression, is difficult to quantify analytically at shorter UV wavelengths. This necessitates the development of a sensitive, precise and accurate colorimetric method for the determination of milnacipran hydrochloride in bulk and swellable matrix tablets. The drug was derivatized with ninhydrin reagent prepared by two methods using direct heating on flame and in glycerol bath at 110°C. The drug -(milnacipran)- ninhydrin complex (Ruhemman’s Purple) showed an absorbance maximum at 570 nm. The method exhibited linearity in a concentration range of 4 to 400 mg/mL with a correlation coefficient of 0.9999. As per ICH guidelines, the method showed good sensitivity (LOD = 0.550 mg/mL and LOQ = 1.667 mg/mL), intraday accuracy and precision (4.713%), and interday accuracy and precision (2.569%). The method was applied to determine the drug in swellable matrix tablets. The obtained results indicate that the proposed method of milnacipran hydrochloride determination in bulk and swellable matrix tablets is simple, accurate and precise, and can be used in less equipped laboratories. Keywords: milnacipran; ninhydrin; Ruhemman’s Purple; colorimetry; LOD; LOQ; matrix tablets.

sion and also has a somewhat superior tolerability compared to TCAs and is similar to selective serotonin reuptake inhibitors [3]. To the best of our knowledge, no official method is available for the quantification of MCN in any pharmacopoeia. Literature review reveals that there are few UV-Vis spectrophotometric techniques for its estimation [4]. A few high performance liquid chromatography (HPLC) methods linked with fluorescence detection are available for its determination in human plasma and some analytical techniques employ chiral HPLC methods [5 – 7]. Some LC/MS methods for MCN determination were also reported [8, 9]. MCN is sensitive to the UV irradiation at shorter wavelength that might be sometimes difficult to judge in dilute samples and samples containing impurities. There is a need to increase the detection wavelength of MCN in order to eliminate the probability of impurities affecting the total concentration of the drug detected at shorter wavelength. In the present work, an attempt was made to derivatize MCN so as to achieve greater detection wavelength in a most common and simplest way possible in a limited facility laboratory, which does not require any sophisticated reagents and instrumentation. MCN with a primary amine present in its

INTRODUCTION Milnacipran hydrochloride (MCN HCl), C15H22N2O×HCl (1R,2S-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropan1-carboxamide hydrochloride) is a new antidepressant characterized by its strong ability to inhibit reuptake of two neurotransmitters (serotonin and norepinephrine reuptake inhibitor, SNRI) with a ratio of 3:1 in favor of norepinephrine (adrenaline) at the synaptic cleft. It is also marked by its virtual absence of affinity to postsynaptic receptors making it highly specific to presynaptic receptor sites [1]. MCN HCl is indicated in the treatment of fibromyalgia in USA and in the treatment of major depression in Europe and Asia [2]. Fibromyalgia is a chronic neurologic widespread muscular pain associated with some general symptoms such as sleep disturbance, fatigue, anxiety, depression, cognitive dysfunction and stiffness. MCN HCl is considered as efficacious as tricyclic antidepressants (TCAs) in management of depres1 2 *

Punjab University College of Pharmacy, University of the Punjab, Allama Iqbal Campus, Lahore-54000, Pakistan. Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore-54000, Pakistan. e-mail: [email protected]

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Simple and Sensitive Colorimetric Method for the Determination of Milnacipran

structure can represent protein compounds that interact with reagents like ninhydrin under optimized reaction conditions, as was previously reported in estimation methods with some analytical compounds [10, 11]. In this study, an effort was initiated to optimize the derivatization conditions so as to develop a simple colorimetric method employing UV-Vis spectrophotometry for MCN determination in parent substance and swellable matrix tablet formulations. EXPERIMENTAL Drug and Chemicals Milnacipran HCl was purchased from Sigma Aldrich, Pakistan. Ninhydrin, hydrochloric acid (HCl), and acetone (Merck, Pakistan) were provided by the University College of Pharmacy, University of the Punjab, Lahore, Pakistan. All reagents and chemicals were of analytical reagent grade. Distilled water was used throughout the experiments. Derivatization of Milnacipran HCl for Colorimetric Analysis Preparation of standard drug solutions. Accurately weighed amount (1 g) of MCN was transferred separately to a volumetric flask of 25 mL capacity containing 10 mL water or HCl buffer pH 1.2 (prepared by mixing 2.982 g potassium chloride and 425 mL 0.2M HCl to make 1000 mL with water) and mixed well until dissolved. The volume was filled to 25 mL with the corresponding solvent (water or HCl buffer). The stock solution of 40 mg/mL was further processed by dilution with water as solvent to the working standards of 400, 80, 32, 24, 16, 12, and 8 mg/mL. Preparation of ninhydrin solutions. Accurately weighed amount of ninhydrin crystals (500, 250, and 200 mg) was separately placed in 100 mL volumetric flasks and dissolved in 50 mL of ethanol. The volume was made 100 mL with ethanol to form working standard solutions (0.5%, 0.25% and 0.2%). Similarly, same quantity of ninhydrin was added to 6 mL acetone and 50 mL water in a 100 mL volumetric flask, dissolved completely, and the volume was made 100 mL with water. Reaction of ninhydrin with MCN. For the purpose of derivatization, 1 mL of the standard drug solutions (standard samples of MCN HCl) covering the wider range of concentrations, i.e., 8 to 400 mg/mL, were added to 10 mL capacity capped test tubes and 20 mL uncapped test tubes already heated to boiling water temperature. Then, 3 mL ninhydrin solution with concentration of 0.2 to 0.5% was transferred to each test tube making the total volume of 4 mL. The capped test tubes were heated in a glycerol bath for 30 min at a temperature of 120 ± 10°C. The uncapped test tubes were heated directly on a uniformly spread naked flame at a distance of 2.5 cm for two minutes to allow the reaction to proceed [12].

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Aliquots (1 mL) of the dark purple colored solution obtained after reaction on naked flame and light purplish brown (reddish) colored solution obtained after the reaction of capped test tubes in glycerol bath were transferred to volumetric flasks for dilution and diluted with distilled water to make the desired concentrations for analysis. The prepared solutions were analyzed by spectrophotometry for absorbance at 570 nm against the blank reagent. Application of Method to Milnacipran Matrix Tablets Accurately weighed matrix tablet containing 100 mg of MCN was crushed and reduced to powder and sieved through mesh 60. The powder was transferred to a conical flask containing 50 mL HCl buffer, pH 1.2. The drug in formulation was extracted by repeated shaking of the flask for sufficient period of time and then filtered to remove the solid polymeric contents. The filtrate was added to 100 mL volumetric flask and made the volume up to 100 mL with aqueous solution. The resulted solution was further diluted to obtain the working sample solution of 80 mg/mL concentration. This solution was reacted with ninhydrin as described above to obtain the purple colored product on naked flame and reddish colored product in glycerol bath. The colored product (Ruhemman’s Purple) was analyzed at 570 nm using UV-Visible spectrophotometer (BMS UV-1602 dual beam, spectrophotometer) and was computed for concentration of MCN in matrix tablets by comparing with the regression equation of the standard curve obtained in derivatization method. Selection of optimum wavelength (l max). Standard solutions of 40, 20, and 8 mg/mL of ninhydrin-derivatized MCN prepared in both solvents (distilled water and HCl buffer pH 1.2) were separately scanned over UV interval of 200 to 600 nm in an UV-Vis spectrophotometer. The spectrum was examined for the maximum absorbance value achieved at the specific wavelength (lmax), which was selected for the subsequent measurement of unknown samples. Construction of calibration curve. The optimized standard drug solution of MCN (400 mg/mL) was derivatized with optimized ninhydrin concentration (0.2% w/v) to form purple blue colored product (naked flame method). Aliquot (2 mL) of the obtained solution was transferred to 10 mL volumetric flask and filled to exact volume using water as solvent (80 mg/mL). This solution was further diluted with water as solvent to working standards of 40, 32, 28, 24, 20, 16, 12, 8, and 4 mg/mL. These concentrations were plotted against the values of absorbance obtained at optimum wavelength 570 nm to construct a calibration curve. All readings were taken in triplicate (Fig. 1).

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Talib Hussain et al. 0.35 0.30 0.25 0.20

y = 0.0033x + 0.0018 R2 = 0.9998

0.15 0.10 0.05 0 Concentration (μg/mL)

Fig. 1. Calibration curve for Ruhemman’s Purple obtained via derivatization of MCN HCl using naked flame method.

Validation of Method The method of MCN HCl quantification was validated according to international guidelines [13, 14]. Multiple parameters with respect to validation were studied, including linearity, accuracy, and precision. The results were also quantified to measure the limit of detection (LOD) and limit of quantitation (LOQ). Linearity. Linearity of the proposed method was established using eleven diluted series of Ruhemman’s Purple solutions of derivatized MCN in distilled water (4 to 80 mg/mL). The dilutions were made from the primary stock solution (400 mg/mL) and analyzed by least squares regression analysis. Accuracy. The accuracy of the proposed method is defined as the proximity of obtained values to the actual values for the dilutions under investigation. The accuracy was determined by selecting three concentrations of derivatized drug solution at lower (4 mg/mL), intermediate (20 mg/mL) and higher (40 mg/mL) concentrations prepared from derivatized stock solution (n = 6). Accuracy was determined in terms of percentage relative error (% error) and mean percentage recovery. Standard addition method to the tablet content derivatized with ninhydrin was also adopted to determine accuracy of the proposed method by adding various concentrations of the standard stock solution (4, 20, 40 mg/mL) to the sample solution (80 mg/mL) and analyzing the total concentration determined by the proposed method (n = 6). Percentage recovery of the standard drug concentration added to tablet content was calculated as %Recovery = [(Ca – Cb)/Cc] × 100%, where Ca is the analyzed total concentration obtained after standard addition to tablet drug concentration, Cb is concentration of the added tablet drug, and Cc is concentration of standard stock solution added to tablet drug solution [15].

Precision. Precision validation of the proposed method consists of the measurement of repeatability and reproducibility of results determined by selecting three different concentrations of derivatized drug substance (same concentrations as in accuracy studies) and analyzed on the basis of intraday and interday variation of results. Different concentrations of the drug prepared in triplicate were analyzed at six different times of day for studies of the intraday variation, and similarly for continuous six days to study the interday variation (n = 6). The results are reported as standard deviation, percentage relative standard deviation (%RSD), percentage error, and percentage recoveries. Similar method was adopted for matrix tablets to assess the applicability of the proposed method to the tablet formulation. Sensitivity. The sensitivity of the proposed method was calculated in terms of LOD and LOQ defined according to ICH guidelines, where LOD is defined as three times of noise of the response signal and LOQ is ten times the noise of the signal. For this determination, different parameters were studied such as the calibration curve and slope of this curve and intercept values of the curve, and then SE and SD of the intercept were calculated. Robustness. The robustness of the proposed derivatized method was calculated by deliberately adopting a small change in parameters to find their effects on the analytical effectiveness of drug determination. The selected parameters for derivatization were 0.2% w/v solution of ninhydrin at a volume of 3 mL (replaced with 0.25% w/v solution) and 1 mL of MCN (400 mg/mL) replaced with 1 mL of 80 mg/mL solution. The absorption maximum was also shifted to a smaller wavelength of 566 nm. The relative standard deviation and recovery were calculated at each time for determining robustness of the proposed method. RESULTS AND DISCUSSION Development of Method Ninhydrin (triketohydrindene hydrate) is primary chromatographic reagent used in the derivatization of many compounds (containing primary amines in their structure) for their quantification purpose. Ninhydrin reacts with primary amines to produce a purple reddish color product known as Ruhemman’s Purple that showed absorption maximum at 570 nm in UV-Vis spectrum. The proposed chemical reaction between ninhydrin and MCN HCl can be represented as depicted in Fig. 2. Optimization of Derivatization Conditions Variables chosen for the conditions of reaction between ninhydrin and MCN HCl were analyzed for their optimization based on the ninhydrin concentration and drug concentration for naked flame and glycerol bath variants and studied to give maximum absorbance at 570 nm.


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Fig. 2. Proposed chemical reaction of MCN HCl with ninhydrin to form Ruhemman’s Purple.

Maximum detection wavelength (lmax). UV spectrum of standard solutions of derivatized MCN (Ruhemman’s Purple) in water and HCl buffer pH 1.2 with concentrations 40, 20, and 4 mg/mL over a range of 200 to 600 nm using UV-Vis spectrophotometer exhibited maximum absorbance at 570 nm. This wavelength was taken as lmax and used to determine Ruhemman’s Purple in all unknown samples in this study. Effect of ninhydrin concentration. The influence of ninhydrin concentration on the reaction was studied for three values (0.2%, 0.25% and 0.5% w/v) with 1 mL of 80 mg/mL MCN HCl solution. It was found that 3 mL 0.2% ninhydrin solution gave maximum absorbance at 570 nm. The ninhydrin concentrations of 200, 250, and 500 mg/mL showed absorbance of 0.335, 0.322 and 0.254, respectively. More concentrated solutions showed lower absorbance or produced weaker purple colored preparation, that was indicative of early completion of reaction at a given concentration and remaining excess ninhydrin solution caused the dilution of sample resulting in decreased absorbance. Effect of MCN HCl concentration. The influence of MCN HCl concentration on the reaction was determined by studying a wider range of solution concentrations (8, 32, 80, 160, and 400 mg/mL) under heating with 3 mL optimized

0.2% w/v ninhydrin solution. It was found that, as the MCN concentration in the reaction mixture increased, the intensity of purple color as well as the absorption maximum at 570 nm in UV-Vis spectrum exhibited proportional growth indicating the optimum concentration of 400 mg/mL. The reaction also indicated the possibility of using all drug concentrations in this reaction with 0.2% w/v ninhydrin concentration. The results are shown in Fig. 3. Effect of heating source and temperature. The influence of heating source selected for the reaction was determined by heating aliquots (1 mL) of optimized 400 mg/mL drug solution with 3 mL of 0.2% w/v ninhydrin solution separately on glycerol bath and naked flame. The results showed that the reaction in glycerol bath at 120 ± 10oC produced reddish purple spots in the reaction mixture and showed fluctuations in the absorbance maximum at 570 nm, which was indicative of incomplete reaction. The reaction mixture also turned yellow on storage for one hour. The absorption at 570 nm vanishes as the mixture color turned yellowish indicating the weakness of reaction separated in the presence of water at room temperature. This could be due to the fact that primary amine of MCN reacted very slowly with ninhydrin, resulting in low color intensity during single reaction time that resulted in incomplete reaction. Therefore, the measure-

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Talib Hussain et al. 1.4

1.6

1.2

1.4

1.0

1.2 1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

Concentration (μg/mL)

Concentration (μg/mL)

Fig. 3. Plot of the absorbance maximum at 570 nm vs. MCN HCl concentration.

ment of color intensity at various times can show if the reaction maximum is achieved [16]. The reaction heated on naked flame showed complete reaction in 2 min of exposure in boiling reaction mixture, which resulted in complete and uniform purple color indicating the formation of Ruhemman’s Purple. Effect of elapsed time on the intensity of reaction. To determine the effect of time elapsed after reaction on the stability of Ruhemman’s Purple when stored at room temperature for a period of 2 h, the reaction mixture reacted on naked flame was allowed to cool down to room temperature. The reaction of MCN HCl at three concentrations (32, 80, and 400 mg/mL) with 0.2% w/v ninhydrin solution was studied for the effect of elapsed time on the intensity of color and subsequent absorption at 570 nm wavelength. The 400 mg/mL concentration produced an intense dark purplish blue color and showed absorption of 1.183 at zero time of cooling after reaction. Similarly, 80 mg/mL showed purplish blue color with 0.274 absorption maximum at 570 nm, and 32 mg/mL reacted with ninhydrin to produce light purple color with 0.101 absorption maximum at 570 nm. The reaction mixture of these three concentrations was first evaluated within 5 min of reaction and then re-evaluated after 60 min and 120 min of the reaction when stored at room temperature and showed stability of the reaction with a mild increase in color intensity as well as the maximum absorption at 570 nm. These results indicated that, MCN (having a primary amino group) reacted with ninhydrin solution in acetone/water media at a temperature of 95 ± 10°C for 50 to 120

Fig. 4. Stability of the absorbance of reaction mixtures with various MCN HCl concentrations stored at room temperature.

sec (~1 to 2 min) remained stable on testing for longer analytical duration required for proper analysis of samples [17]. The results are summarized in Fig. 4. Validation of the Developed Method The utility of analytical methods that are easy and do not require high-efficiency, expensive and time consuming instrument is always encouraged in pharmaceutical analysis. The UV-Vis spectrophotometry method for determination of drugs for analysis purpose is considered most important in this respect. The following parameters were studied to establish the validity of the developed method. Linearity. A linear calibration curve was obtained over the range of 4 to 80 mg/mL concentrations prepared from the primary derivatized 400 mg/mL concentration of Ruhemman’s Purple with correlation coefficient obtained greater than 0.999. The best fit linear regression equation was y = 0.0033x + 0.0018, R2 = 0.9998 (Fig. 1). The results showed excellent correlation between the absorbance values obtained against the concentrations implied in this study. The calculated standard deviation values of determined absorbance against concentrations were found considerably low and RSD values were below 3.5%. The absorbance range was within 0.0154 to 0.254 for 4 to 80 mg/mL and 1.125 for maximum concentration of 400 mg/mL and linearity was maintained even for computation of maximum concentrations in the calibration curve.

TABLE 1. MCN Recovery in Swellable Matrix Tablets (n = 3) Tablet sample solution (mg/mL) Cb

Standard solution (mg/mL) Cc

Total conc. (mg/mL) Ca

Recovery (%) ± SD

% RSD.

% Error

80

4

84

101.894 ± 0.001

9.12

1.89

80

20

100

99.924 ± 0.0025

5.28

0.08

80

40

120

101.856 ± 0.0038

4.06

1.86


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TABLE 2a. Intraday Precision Analysis of MCN HCl Derivative (n = 6) Sr. No.

Standard solution (mg/mL)

Found conc. ± SD (mg/mL)

Recovery (%)

% RSD

% Error

1

4

4.088 ± 0.0005

102.19

5.34

2.19

2

20

20.14 ± 0.0019

100.70

4.83

0.70

3

40

40.49 ± 0.0031

101.23

3.96

1.22

TABLE 2b. Interday Precision Analysis of MCN HCl Derivative (n = 6) Sr. No.

Standard solution (mg/mL)


Recovery (%)

% RSD

% Error

1

4

4.092 ± 0.0004

102.31

4.99

2.31

2

20

20.30 ± 0.0005

101.48

1.38

1.48

3

40

40.76 ± 0.0009

101.90

1.33

1.89

Accuracy. The error and percentage recovery of the three concentrations of derivatized MCN analyzed were determined to find the accuracy of the method. The values of standard deviation, relative standard deviation, percentage recovery and error are summarized in Table 1. To establish accuracy of the method, standard addition method to 80 mg/mL tablet concentration at three varying concentrations (4, 20, and 40 mg/mL) was analyzed for recovery and appropriate results within acceptable limits were found for both recovery error calculations. Precision. The reproducibility and repeatability of the method were determined by analyzing the concentration of derivatized MCN at six different time intervals on the same day in triplicate as well as over six different days with fresh derivatization every day in triplicate. The data on interday (Table 2a) and intraday (Table 2b) precision are demonstrated in terms of percentage RSD along with percentage recovery, standard deviation, and error. The results of precision analysis indicated that the proposed method is more sensitive for lower as well as higher concentrations of derivatized MCN. Sensitivity. The sensitivity of the proposed method was determined as LOD that was found to be 0.550 mg/mL and LOQ determined as 1.667 mg/mL. These values demonstrate that the method is sufficient to detect as well as quantify the

minimum amount of MCN in matrix tablets with good sensitivity. The sensitivity of the method proved to be in conjunction or even superior to the UV detection methods and HPLC determinations developed for MCN HCl [10, 18, 19]. Robustness. The analysis of deliberate change in the derivatization parameters indicated that there was a minor change in analytical results with similar derivatized color and absorption values. The percentage recovery and standard deviation were calculated every time and were found to be 100.12% and 0.00821, respectively. The percentage RSD was found to be 2.36%. The results indicated that minor changes were expected to affect the analytical performance, but it did not adversely affect the analytical procedure showing excellent sensitivity and robustness of the proposed method. A summary of validation parameters representing optical and regression characteristics are given in Table 4. Analysis of MCN HCl swellable matrix tablets Two formulations of MCN HCl (M21 and M24) prepared by homogeneously blending 100 mg of MCN HCl with chitosan (low and medium molecular weight) and polycaprolactone with two solvents (dichloromethane and chloroform respectively), were analyzed by the developed method (n = 3) for the drug content. The formulation codes

TABLE 3. Assay of MCN HCl in Swellable Matrix Tablets Sr. No.

Extracted drug conc. from tablets (mg/mL)


Recovery (%)

% RSD

% Error

1

80

79.99 ± 0.0051

99.98

2.030

0.013

2

80

79.47 ± 0.0062

99.33

2.488

0.664

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Talib Hussain et al.

TABLE 4. Optical and Regression Characteristics of the Proposed Method Parameter

lmax, nm

Value

REFERENCES

570

Beer’s law limit, mg/mL

4 – 400

Molar absorptivity, L/(mol cm) Regression equation

3.0547 ´ 10

3

y = 0.0031x + 0.0024

Slope ± SD

0.0031 ± 0.00019

Intercept ± SD

0.0028 ± 0.00051 2

drug concentration. The sample recovery studies were close to the claimed amounts of MCN HCl in tablets.

Correlation coefficient (R )

0.9999

Limit of detection (LOD), mg/mL

0.550

Limit of quantitation (LOQ), mg/mL

1.667

Intraday precision (% RSD)

4.713

Interday precision (% RSD)

2.569

were designed according to parallel formulation development study where M stands for MCN and subscripts 21 and 24 indicate the order of preparation in same study according to the method stated above (under publication). Good results of determination of the drug content suggest the method to be efficient. The results of assay of MCN swellable matrix tablets are given in Table 3. CONCLUSION The colorimetric method developed for MCN HCl analysis by UV-Vis spectrophotometry is simple, sensitive, precise and accurate. This method can be applied to common assessment of raw drug and routine analysis of pharmaceutical dosage forms in quality control as evident from recovery studies employed. The wide range of detection (4 to 400 mm/mL) and very low LOD (0.550 mg/mL) and LOQ (1.667 mg/mL) show the method to be sensitive and quantitative to very low

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