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A comparative study of smart spectrophotometric methods for simultaneous determination of a skeletal muscle relaxant and an analgesic in combined dosage form
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 166–173
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A comparative study of smart spectrophotometric methods for simultaneous determination of a skeletal muscle relaxant and an analgesic in combined dosage form Hesham Salem c, Dalia Mohamed a,b,⇑ a b c
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, 11787 6 October City, Egypt Analytical Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, 11795 Cairo, Egypt Analytical Chemistry Department, Faculty of Pharmacy, Deraya University, 14511 Cairo, Egypt
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Six different spectrophotometric
methods based on isosbestic point or ratio spectra. All methods could be applied for simultaneous analysis of PARA and DANT. All methods could be easily applied in QC labs. No significant difference in accuracy and precision compared to reported method. They are applied for analysis of the dosage form without interference of excipients.
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 5 December 2014 Accepted 28 December 2014 Available online 3 January 2015 Keywords: Absorbance subtraction method Amplitude modulation method Dantrelax compoundÒ capsules Dantrolene sodium Paracetamol
a b s t r a c t Six simple, specific, accurate and precise spectrophotometric methods were developed and validated for the simultaneous determination of the analgesic drug; paracetamol (PARA) and the skeletal muscle relaxant; dantrolene sodium (DANT). Three methods are manipulating ratio spectra namely; ratio difference (RD), ratio subtraction (RS) and mean centering (MC). The other three methods are utilizing the isoabsorptive point either at zero order namely; absorbance ratio (AR) and absorbance subtraction (AS) or at ratio spectrum namely; amplitude modulation (AM). The proposed spectrophotometric procedures do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined. The selectivity of the developed methods was investigated by analyzing laboratory prepared mixtures of the drugs and their combined dosage form. Standard deviation values are less than 1.5 in the assay of raw materials and capsules. The obtained results were statistically compared with each other and with those of reported spectrophotometric ones. The comparison showed that there is no significant difference between the proposed methods and the reported methods regarding both accuracy and precision. Ó 2015 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author at: Analytical Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, 11795 Cairo, Egypt. Mobile: +20 1000664243. E-mail address: [email protected] (D. Mohamed). http://dx.doi.org/10.1016/j.saa.2014.12.099 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Paracetamol (PARA); N-acetyl-p-aminophenol; (Fig. 1a) is a widely used analgesic and antipyretic agent for the relief of fever, headaches and minor pains. It is a major ingredient in numerous cold and flu remedies. In combination with non-steroidal anti-
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inflammatory drugs and opioid analgesics; paracetamol is also used in the management of severe pain (such as post-operative pain) [1,2]. Paracetamol alone or in combination with other drugs was estimated by titrimetry [3,4], spectrophotometric method [5– 7], HPLC [8,9], TLC [10], HPTLC [11], UHPLC [12], LC–MS [13], FT-IR [14], amperometric determination [15] and fluorimetry [16]. Dantrolene sodium (DANT); 1-[5-(4-nitrophenyl)furfurylidene amino]imidazolidine-2,4-dione sodium salt (Fig. 1b) is a muscle relaxant with a direct action on skeletal muscles [1]. DANT uncouples muscular contraction from excitation, probably by interfering with the release of calcium from the sarcoplasmic reticulum [1]. DANT was also determined either alone, in presence of its metabolites and impurities or in biological fluids utilizing different methods as HPLC [17–20] and polarography [21]. Most of the drugs used for treatment of skeletal muscle disorders are combined with analgesics such as PARA. PARA and DANT are formulated as capsule where this combination is used for myalgia, sprains and skeletal muscle spasms due to neurological disorder. Literature search revealed only two methods for the simultaneous determination of PARA and DANT in dosage form; HPLC-UV [22] and spectrophotometry based on zero order, first derivative and derivative ratio methods [23]. The main problem of spectrophotometric binary or ternary mixture analysis is the simultaneous determination of the compounds in the same mixture without prior separation. Several spectrophotometric methods have been used for resolving such mixtures with overlapped spectra such as; solving two simultaneous equations (Vierodet’s method) [24], Derivative spectrophotometry [25], ratio derivative spectrophotometry [26,27], in addition to, H-point standard addition method [28]. Thus, the aim of this work was to develop and conduct comparative study on recently developed well established spectrophotometric methods based either on ratio spectra (RD, RS, MC) or on isosbestic point (AR, AS and AM) for resolving binary mixture of spectral interfering problem without preliminary separation. The utilized methods are very simple, accurate, precise and do not require any sophisticated apparatus. Theory Absorbance ratio method (AR) Erk [29] developed a spectrophotometric method which is based on the linear relationship between the absorbance ratio value of a binary mixture of X and Y and the relative concentration of such a mixture. The main criteria for the application of this method are the existence of an isoabsorptive point and the selection of optimum wavelengths (k1, k2, kiso) which can fulfill good determination of the components in the binary mixture. Absorbance subtraction method (AS) This method is based on the same principles as the absorbance factor method [30–33]. However, this method could be applied for
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the analysis of a mixture of two drugs X and Y having overlapped spectra intersecting at isoabsorptive point where Y is extended over X, and X doesn’t show any contribution at another wavelength (k2). In this method, the isoabsorptive point kiso could be used for separate quantitative estimation of each X and Y in their mixture (X + Y). The determination can be done using mathematically calculated factor of one of these components. By simple manipulation step, we can obtain the absorbance value corresponding to X and Y, separately. So, the concentration of each component could be obtained via the isoabsorptive point regression equation without any need for a complementary method. Amplitude modulation method (AM) The method is a ratio spectrum manipulating method using the normalized spectrum of the divisor obtained by dividing certain spectrum of Y0 component by its concentration [30,32]. For a mixture of X and Y, where Y is more extended than X; X and Y shows isoabsorptive point at the zero order spectrum and consequently is retained as an isosbestic point at the ratio spectrum. Experimental Apparatus and software A JASCO V-530 double beam UV–VIS spectrophotometer loaded with Spectra Manager Program (JASCO) was used for spectral acquisition and elaboration of the data obtained. Quartz cuvettes, 1-cm pathlength were used for measuring the light absorption in the ultraviolet region (200–400 nm). For the mean centering for ratio spectra method, MatlabÒ Version 7.9 was used. Chemicals Pure samples: paracetamol (PARA) was kindly supplied by International Drug Agency for Pharmaceutical Industry (Portsaid, Egypt). Dantrolene sodium (DANT) was kindly supplied by Chemipharm Pharmaceutical Industries (6th October City, Egypt). Their purity was found to be 99.82 ± 0.71 and 99.83 ± 0.76 for PARA and DANT, respectively, by a reported method [23]. Market sample: Dantrelax compoundÒ capsules labeled to contain 300 mg PARA and 25 mg DANT (Batch number: 131186A), was manufactured by Chemipharm Pharmaceutical Industries (6th October City, Egypt). Solvents: methanol was obtained from Merck (Darmstadt, Germany). Standard solutions Separate stock solutions of PARA and DANT (1 mg/mL) were prepared by dissolving the compounds in methanol then completing in 100 mL measuring flasks. The working solutions were freshly prepared by dilution from the stock solutions with the same solvent to obtain a concentration of (0.1 mg/mL) for each of PARA and DANT. Procedures Spectral characteristics The zero-order absorption spectra (D0) of the two components were recorded at (200–400 nm) against methanol as a blank.
Fig. 1. Chemical structures of (a) paracetamol (PARA), (b) dantrolene sodium (DANT).
Construction of calibration curves Ratio difference method (RD). Standard solutions containing 1–30 lg/mL PARA and 1–40 lg/mL DANT were prepared separately in methanol. The absorption spectra of the resulting
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solutions were measured and divided by the absorption spectra of 25 lg/mL DANT and 20 lg/mL PARA, where the obtained ratio spectra were recorded. Construct calibration curves for PARA and DANT by plotting the difference between the amplitudes of the obtained ratio spectra at 253 and 218 nm and 218 and 253 nm respectively, versus the corresponding concentrations. The regression equations were computed. Ratio subtraction method (RS). Standard solutions of PARA containing 1–30 lg/mL were prepared in methanol. The absorption spectra of the resulting solutions were measured. Construct calibration curves relating the zero order spectra of PARA at 248 nm versus the corresponding concentrations and compute the regression equations. Mean centering method (MC). Standard solutions containing 1– 30 lg/mL PARA and 1–40 lg/mL DANT were prepared separately in methanol. The absorption spectra of the resulting solutions were measured and divided by the absorption spectra of 25 lg/mL DANT and 20 lg/mL PARA, where the obtained ratio spectra were recorded and then mean centered. Construct calibration curves relating the amplitudes of the mean centered values to the corresponding concentrations at 253 nm for PARA and 218 nm for DANT. Absorbance ratio method (AR). Separate standard solutions of PARA and DANT each containing 1–30 lg/mL were prepared separately in methanol. The absorption spectra of the resulting solutions were measured. Construct calibration curves relating the zero order spectra of PARA at 248 nm and DANT at 380 nm versus the corresponding concentrations and compute the regression equations. Absorbance subtraction method (AS). Separate standard solutions of PARA and DANT each containing 1–30 lg/mL were prepared separately in methanol. The absorption spectra of the resulting solutions were measured. Construct calibration curves relating the zero order spectra of PARA or DANT at 274 nm versus the corresponding concentrations and compute the regression equations. The absorbance factor of DANT [A274/A380] was calculated and was found to be 0.27. Amplitude modulation method (AM). Separate standard solutions of PARA and DANT each containing 1–30 lg/mL were prepared separately in methanol. The absorption spectra of the resulting solutions were measured and divided by the normalized divisor of DANT spectrum, and the obtained ratio spectra were recorded. The calibration curves were constructed by plotting the amplitudes of PARA or DANT at 274 nm against their corresponding concentrations and the regression equations were computed. Application to laboratory prepared mixtures Into a series of 10-mL volumetric flask, accurate aliquots of PARA and DANT were transferred from their working solutions to prepare seven mixtures containing different ratios of the cited drugs. The volumes were completed with methanol. The spectra of the prepared solutions were recorded at (200–400 nm). The concentration of each drug was calculated by substitution in the corresponding regression equation after applying the corresponding manipulating steps for each method. In case of ratio subtraction method, the mixtures were divided by 25 lg/mL DANT as a divisor followed by subtraction of the obtained constant from the division at the plateau (310–380 nm), then multiply the obtained spectrum by the spectrum of 25 lg/mL of DANT. While in case of absorbance ratio method; the analysis of PARA and DANT in binary mixture was performed by using the following equations:
C 1 ¼ ðQ 1 b1 Þ=a1 ðAiso =aiso Þ C 2 ¼ ðQ 2 b2 Þ=a2 ðAiso =aiso Þ where Q1 = A1/Aiso for PARA, Q2 = A2/Aiso DANT. C1, C2 = concentrations of PARA and DANT, respectively. Aiso = absorbance at isoabsorptive point, where kiso = 274 nm. aiso = absorptivity at isoabsorptive point = Aiso/C1 + C2, a1 = slope of regression equation (Q1 versus C1/C1 + C2), a2 = slope of regression equation (Q2 versus C2/C1 + C2), b1, b2 = intercept values for these regression equations. A1 and A2 denote the absorbances of the mixture solution measured at k1 (248 nm) and k2 (380 nm). Application to pharmaceutical preparation Ten capsules were evacuated and the content was well mixed. An accurate amount of the powder equivalent to 120 mg PARA and 10 mg DANT was weighed and transferred into a beaker, 50 mL methanol were added with continuous stirring for about 10 min. The solution was filtered into 100-mL volumetric flask. The volume was completed with methanol. Aliquots equivalent to 0.15 mL from this solution were transferred to 10-mL volumetric flasks and the volume was completed with methanol to obtain a final concentration of 18 lg/mL for PARA and 1.5 lg/mL for DANT. The concentration of each drug was calculated using the corresponding regression equation. When carrying out the standard addition technique, different known concentrations of pure standard of each drug were added to the pharmaceutical dosage form before proceeding in the previously mentioned methods. Results and discussion The use of spectrophotometric methods such as derivative spectrophotometry, ratio spectra spectrophotometry and other chemometric spectral calibration techniques is crucial for analytical studies, related to the quality control and routine analysis of commercial products in the research or industry laboratories. It was found that these spectrophotometric methods are preferable instead of hyphenated analytical instrumentations or techniques such as LC–MS, GC–MS and LC–NMR which always require prior extraction and separation steps and other tedious analytical process during analysis. On the other hand, the related techniques having complex components bring high cost and time consumption. Taking into account all above arguments, the quantitative spectrophotometric resolution of the mixtures of two or more compounds having overlapped spectra is an interesting issue for the analytical chemistry. Besides, the existing spectrophotometric methods were found to be very easy to apply, very rapid, sensitive and yet very cheap for analysis of mixture. Fig. 2 shows the overlapping zero-order absorption spectra (D0) of PARA and DANT. Although DANT can be determined at its kmax at 380 nm, however, this does not provide simultaneous determination of both drugs. In addition DANT shows high absorbance at 248 nm hindering the determination of PARA at its kmax (248 nm). Thus, the main task of this work was to establish simple, sensitive and accurate spectrophotometric methods for the simultaneous determination of paracetamol and dantrolene sodium in their bulk powders and pharmaceutical dosage form with satisfactory precision for good analytical practice. In addition, to construct a statistical comparison between the ability of the proposed methods in determining the two drugs. Ratio difference method (RD) The overlapped spectra of PARA and DANT suggested that ratio difference spectrophotometric method is as a suitable method for their simultaneous determination. Lotfy and Hegazy [34]
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Fig. 2. Absorption spectra of 20 lg/mL PARA (_____), 20 lg/mL DANT (— – — –) and a binary mixture of 10 lg/mL of each PARA and DANT (– – – –).
introduced the ratio difference spectrophotometric method (RD) which depends on measuring the difference between two amplitude values at two wavelengths in one step using the ratio spectra, so it eliminates the derivative steps and therefore the signal to noise ratio is enhanced [35–37]. The linearity of the amplitude values at each of the chosen wavelength against the corresponding concentration should be checked to ensure minimal noise to signal ratio at these wavelengths. By applying the ratio difference method, the difference between the amplitude values is measured by subtraction the two values at two selected wavelength, so the noise will be cancelled. For the determination of PARA concentration by this method, the only requirement was the contribution of PARA and DANT at the two selected wavelengths (253 and 218 nm) where the ratio spectrum of DANT showed the same amplitudes (constant) whereas PARA showed significant difference in these two amplitude values at these two selected wavelengths with concentration. Similarly, DANT could be estimated at the selected wavelengths (218 and 253 nm). PARA (1–30 lg/mL) spectra were divided by the spectrum of DANT (25 lg/mL), Fig. 3a, and DANT (1–40 lg/mL) spectra were divided by the spectrum of PARA (20 lg/mL), Fig. 3b. The concentration of PARA was calculated by using the regression equation representing the linear relationship between the differences of these ratio spectra amplitudes at the two selected wavelengths versus the corresponding concentration of the drug. Similarly, DANT could be determined in the same manner. Different concentrations of PARA (10, 20 and 25 lg/mL) and of DANT (15, 25 and 35 lg/mL) were tested as a divisor but the concentrations 20 lg/mL of PARA and 25 lg/mL of DANT gave minimum noise, smoother ratio spectra and maximum sensitivity. The selected wavelengths were the best regarding average recovery percent when used for the prediction of PARA and DANT concentrations in bulk powder as well as in laboratory prepared mixtures. Ratio subtraction method (RS) The ratio subtraction method (RS) [38] was used for the determination of PARA concentration in the binary mixture of PARA and DANT. The mixture’s spectrum is divided by using certain concentration of one component as a divisor, where the constant value is calculated along the plateau region which is parallel to the baseline, then subtracted from the ratio spectra followed by multiplication by the divisor. In ratio subtraction the component of interest is recovered in its zero order spectra and measured at its kmax to minimize the noise error.
Fig. 3. Ratio spectra of: (a) PARA (1–30 lg/mL) and 25 lg/mL DANT (- - - -) using 25 lg/mL DANT as a divisor and (b) DANT (1–40 lg/mL) and 20 lg/mL PARA (- - - -) using 20 lg/mL PARA as a divisor, both spectra showing the two selected wavelengths (218 and 253 nm).
Thus, PARA can be determined by dividing the spectrum of the binary mixture by a known concentration of DANT as a divisor (25 lg/mL). The constant obtained from the division was measured at the plateau (310–380 nm). By subtracting this constant value, then multiply the obtained curve after subtraction by the spectrum of 25 lg/mL of DANT (the divisor), therefore we can recover the zero order absorption spectrum of PARA present in the mixture, Fig. 4, and measured at its kmax (248 nm). Mean centering method (MC) The obtained absorption spectra of the mixture are divided by the spectrum of standard solution of one component and then mean centered (Fig. 5). PARA has been determined at 253 nm in a concentration range of 1–30 lg/mL, using the spectrum of DANT (25 lg/mL) as a divisor, while, DANT was determined at 218 nm in a concentration range 1–40 lg/mL, using the spectrum of PARA (20 lg/mL) as a divisor. The careful choice of the divisor and the working wavelengths were of great importance, consequently different concentrations of PARA (10, 20 and 25 lg/mL) and of DANT (15, 25 and 35 lg/mL) were tried as divisors. It was found that concentrations 20 lg/mL of PARA and 25 lg/mL of DANT gave minimum noise and better selectivity. Absorbance ratio method (AR) The absorbance ratio method was applied to determine the concentration of PARA (C1) and DANT (C2) in a binary mixture through the linear relationship between the absorbance ratio value of the binary mixture of PARA/DANT and the relative concentration in that mixture. The main criteria for the application of the absorbance ratio method are the existence of an isoabsorpative point and selection of optimum wavelengths. For good separation, the
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Fig. 5. Mean centered ratio spectra of: (a) PARA (1–30 lg/mL) using 25 lg/mL DANT as a divisor and (b) DANT (1–40 lg/mL) using 20 lg/mL PARA as a divisor.
Fig. 4. (a) Division spectra of laboratory prepared mixtures using 25 lg/mL DANT as a divisor, (b) division spectra after subtraction of the constants, (c) the obtained zero order spectra of PARA after subtraction of constant and multiplication by the spectrum of 25 lg/mL DANT.
absorbance of PARA was obtained by subtraction. The absorbance values of PARA and DANT at kiso 274 nm were used to calculate each of their concentration using the unified regression equation at the same wavelength.
ADANT at 274 nm ¼ ½A274 =A380 : ðAmix at 380 nmÞ APARA at 274 nm ¼ Amix at 274 nm ADANT at 274 nm:
method should be applied using the wavelengths (k1, k2, kiso) which can fulfill good determination of the components in the binary mixture [29]. The choice of the two wavelengths (248 and 380 nm) beside the isoabsorpative point (274 nm) gave the best recovery percentages for determination of laboratory prepared mixtures, (Fig. 2). The absorbance of the previous laboratory prepared mixtures at kiso (274 nm) were plotted against total concentrations of (C1) and (C2) and the regression equation was computed, to obtain slope of the regression line (aiso = 0.0174). The absorbance of each solution was measured at k1 (248 nm), k2 (380 nm) and kiso (274 nm), the relative absorbance (Q1 = A1/Aiso) were plotted against the relative concentration (C1/C1 + C2) and the regression equation parameters were computed (b1/a1 = 0.257). The relative absorbance (Q2 = A2/Aiso) were plotted against the relative concentration (C2/C1 + C2) and the regression equation parameters were computed (b2/a2 = 0.0041).
Absorbance subtraction method (AS) This method is based on the isosbestic point present in zero order absorption spectra known as the isoabsorptive point, where the components exhibiting this point have equal absorptivities. For the determination of PARA and DANT, their isosbestic point at 274 nm was utilized, Fig. 2. The absorbance of DANT at 274 nm was calculated using its absorbance factor at this point (where [A274/A380] for DANT was found to be 0.27), and then the
where Amix is the absorbance of the binary mixture, ADANT and APARA are the absorbance of DANT and PARA, respectively; and [A274/A380] is the absorbance factor of pure DANT at 274 nm to that at 380 nm. The requirements of this method are the existence of isoabsorptive point of both components and the extension of the spectra of one component. The advantage of the absorbance subtraction method over the conventional isoabsorpative point is that there is no need for a complementary spectrophotometric method to measure the concentration of one of the components. Both components can be determined using unified regression equation at kiso. The disadvantage of this method is the increased risk of error in calculating the absorbance factor in case of low concentrations of the extended component (DANT) or its low value of absorbance at extension region. Amplitude modulation method (AM) This proposed method is based on two facts; foremost the isosbestic point whenever present in an absorption spectrum will be retained at the same point even after division by one component as a divisor in the ratio spectrum. As known the results of manipulating ratio spectra techniques are greatly affected by the choice of the divisor. Thus, to eliminate the effect of the divisor, the normalized spectrum of DANT was used. By dividing the spectrum of the binary mixture by the normalized DANT divisor spectrum, we obtained the ratio spectra, Fig. 6. The amplitude value of the constant was determined at the plateau region at (310–380 nm), which was equal to the amplitude of the
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Linearity and range The linearity of the proposed methods was evaluated by processing the different calibration curves on three different days. The calibration curves were constructed within concentration ranges that were selected on the basis of the anticipated drugs concentration during the assay of the dosage form. The linearity of the calibration curves were validated by the high value of correlation coefficients. The analytical data of the calibration curve including concentration ranges and calibration equations are summarized in Table 1. Accuracy
Fig. 6. Ratio spectra of a mixture of 20 lg/mL of each PARA and DANT using normalized DANT divisor after subtraction of the constant.
constant of DANT along the whole spectrum. At the isosbestic point (kiso) at 274 nm, the amplitude of the ratio spectra at this point was equal to the sum of the amplitudes of DANT and PARA. By applying ratio manipulation using the normalized divisor, the curve of interfering substance (DANT) was converted to a constant, which is a straight line parallel to wavelength axis having the same amplitude, so there was no need for calculation of response factor. By subtracting the previously obtained constant, we obtained the corresponding amplitude of PARA, which was equivalent to recorded concentration of PARA in the mixture (CRecorded of PARA). To eliminate any error due to signal to noise ratio, the actual concentration of DANT or PARA were calculated by using their corresponding unified regression equation at kiso 274 nm.
C Recorded ¼ 0:9999C þ 0:0186: where CRecorded represents the recorded amplitude of ratio spectrum which was equal to the recorded concentration of PARA and C represented corresponding concentration of PARA. The requirements of this method are the existence of isoabsorptive point of both components at zero order spectrum and consequently in the ratio spectra; and the extension of the spectra of one component. The advantage of amplitude modulation method over other mathematical techniques utilizing the constant is the reduced manipulation steps and only one divisor is needed in order to determine both components in the mixture. By using the normalized divisor, the results are not affected by the choice of divisor and it minimizes the noise in the obtained ratio spectra, where the recorded amplitudes are directly modulated to concentrations in order to minimize the calculation steps. This divisor was obtained by dividing certain spectrum of DANT by its concentration or by dividing the addition of several spectrum of DANT by the sum of their corresponding concentrations. This method has advantage over the isoabsorpative point at zero order that it measures the concentration of both components with no need for other conventional method to measure one of the components in the mixture. This method has advantages over the newly developed absorbance subtraction method (AS) through using the normalized divisor, where the obtained amplitude at the ratio spectrum will directly represent the concentration of each component and so the risk of error upon the determination of absorbance factor of lower absorbance will be reduced through the elimination of the absorbance factor step. Methods validation Methods validation was performed according to ICH guidelines [39] for the proposed methods as follows:
To study the accuracy of the proposed methods, procedures under construction of the calibration curves, for both drugs using the proposed methods, were repeated three times for the determination of seven different concentrations of pure PARA and DANT within the linearity range. The accuracy expressed as percentage recoveries (mean) and standard deviation was shown in Table 1. Good accuracy of the developed methods was indicated by the results obtained. Precision The intra-day and inter-day precision of the proposed methods were determined by the analysis of three different concentrations of pure PARA and DANT, within the linearity range, by three replicate analyses of three pure samples of both drugs on a single day and on three consecutive days, respectively. The results were illustrated in Table 1. Selectivity Selectivity was ascertained by analyzing different mixtures containing both drugs in different ratios within the linearity range. Satisfactory results were shown in Table 2. Good recovery percentages with accepted standard deviations were obtained in all cases. Limits of detection and quantification The LOD is the lowest concentration of the drug that can be detected, but not necessarily quantitated, under the stated experimental conditions, while, the LOQ is the lowest concentration of the analyte that, can be determined with acceptable precision and accuracy. Both LOD and LOQ were calculated and the results are abridged in Table 1. Application of the proposed methods The proposed procedures were applied for the determination of PARA and DANT in Dantrelax compoundÒ capsules where the obtained recovery and standard deviation proved no excipients’ interference. The validity of the proposed procedures is further assessed by applying the standard addition technique. The results obtained are shown in Table 3. Statistical analysis Table 4 showed statistical comparisons of the results obtained by the proposed methods and reported method [23]. The calculated t and F values were less than the theoretical ones indicating that there was no significant difference between the proposed and the reported methods with respect to accuracy and precision. Oneway ANOVA was applied for the purpose of comparison of devel-
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Table 1 Assay parameters and validation sheet obtained by applying the proposed spectrophotometric methods. Parameter
RD
Wavelength (nm) Calibration rangea (lg/mL) Slope Intercept Correlation coefficient Mean RSD% LOD LOQ Repeatabilityab Inter-day precisionab a b
RS
MC
AR
AS
AM
PARA
DANT
PARA
PARA
DANT
PARA
DANT
PARA
P1–P2 253–218 1–30 0.1669 0.0165 0.9997 99.77 0.67 0.19 0.58 0.61 0.75
P1–P2 218–253 1–40 0.0556 0.0414 0.9997 99.46 0.77 0.18 0.55 0.59 0.79
D0 at 248 1–30 0.0857 0.005 0.9998 100.46 0.50 0.21 0.64 0.65 0.71
MC at 253 1–30 0.122 0.0159 0.9996 99.77 0.69 0.28 0.85 0.71 0.85
MC at 218 1–40 0.0391 0.0413 0.9997 99.45 0.78 0.23 0.70 0.62 0.65
D0 at 248 1–30 0.0857 0.005 0.9998 100.46 0.50 0.21 0.64 0.65 0.71
D0 at 380 1–30 0.0655 0.0108 0.9997 100.39 0.64 0.23 0.70 0.69 0.84
D0 at 274 1–30 0.0174 0.0040 0.9999 100.00 99.90 0.66 0.67 0.18 0.55 0.74 0.65 0.79 0.82
DANT
PARA
DANT
P at 274 1–30 0.9999 0.0186 1.000 99.99 99.98 0.88 0.54 0.30 0.91 0.77 0.65 0.94 0.81
Average of three experiments. Relative standard deviation of three concentrations of each drug (5, 15, 25 lg/mL).
Table 2 Analysis of laboratory prepared mixtures by the proposed spectrophotometric methods. Conc in lg/mL PARA:DANT
10:20 5:25 12:1b 10:10 20:10 25:5 24:2b Mean RSD%a SEMc Variance a b c
RS
MC
PARA
RD DANT
PARA
PARA
DANT
AR PARA
DANT
AS PARA
DANT
AM PARA
DANT
98.64 98.22 98.09 100.40 99.12 98.25 100.19 98.99 0.97 0.37 0.9227
101.42 103.05 100.29 101.35 103.44 102.25 101.50 101.90 1.07 0.40 1.1838
99.79 100.55 100.35 98.63 98.76 98.92 99.27 99.47 0.78 0.29 0.5970
99.90 99.21 99.65 100.22 98.63 98.30 99.96 99.41 0.73 0.27 0.6557
99.79 100.71 100.45 100.36 100.63 100.53 98.94 100.20 0.63 0.24 0.6087
101.27 99.26 100.05 98.82 101.16 100.80 99.46 100.12 0.97 0.37 0.9514
100.30 98.72 100.04 100.60 98.76 99.17 98.71 99.47 0.82 0.31 0.6697
99.90 99.50 100.72 100.15 98.51 98.91 99.46 99.59 0.75 0.28 0.5567
99.12 100.99 99.35 100.12 99.95 99.12 98.38 99.58 0.85 0.32 0.7225
97.60 98.40 99.83 98.70 98.60 98.24 99.20 98.65 0.72 0.27 0.5068
100.88 100.02 102.00 100.02 99.99 101.20 101.40 100.79 0.79 0.30 0.6400
Percent relative standard deviation. Ratio present in dosage form. SEM is the standard error of the mean.
Table 3 Application of standard addition technique to the analysis of dosage form by applying the proposed spectrophotometric methods. Methods
RD RS MC AR AS AM a b c
PARA
DANT
Found in lg/mLa
b
17.76 18.14 17.90 17.91 18.00 17.91
98.69 ± 0.64 100.77 ± 1.00 99.46 ± 0.77 99.52 ± 0.75 100.00 ± 0.59 99.50 ± 1.07
Recovery % ± SD
Pure addedc recovery % ± SD
Found in lg/mLa
b
Pure addedc recovery % ± SD
98.44 ± 1.05 100.82 ± 1.07 99.36 ± 0.84 99.37 ± 0.92 99.87 ± 0.77 100.13 ± 0.91
1.52
101.60 ± 1.10
101.25 ± 1.03
1.51 1.50 1.49 1.51
100.51 ± 0.76 100.00 ± 0.87 99.33 ± 0.87 100.53 ± 0.86
100.10 ± 0.89 99.35 ± 0.54 99.61 ± 0.96 99.92 ± 0.72
Recovery % ± SD
PARA claimed to be 18 lg/mL, DANT claimed to be 1.5 lg/mL. Average of five experiments. Average of three experiments (added equivalent to 2, 4, 6, 8, 12 lg/mL PARA and 0.5, 1, 3.5, 8.5, 20.5 lg/mL DANT).
oped methods; where Table 5 shows no significant difference between the developed methods. Conclusion This work describes six simple, specific, accurate and precise spectrophotometric methods for the simultaneous determination of paracetamol and dantrolene sodium in bulk powder, in laboratory prepared mixture and in dosage form. Three of the performed methods namely; absorbance ratio, absorbance subtraction and amplitude modulation have the ability to analyze the two drugs simultaneously and all require the presence of isoabsorptive point. Absorbance ratio has the advantage that spectral measurements are done at zero order which allows maximum accuracy and reproducibility but unfortunately it requires multiple calculation steps.
Absorbance subtraction allows the determination of the two interfering components using unified regression equation at kiso. However, the disadvantage of this method is the increased risk of error in calculating the absorbance factor in case of low concentrations of the extended component. Amplitude modulation has advantages over the newly developed absorbance subtraction method through using the normalized divisor, where the obtained amplitude at the ratio spectrum will directly represent the concentration of each component and so the risk of error upon the determination of absorbance factor of lower absorbance will be reduced through the elimination of the absorbance factor step. In addition to the previous methods, three other methods were performed in which ratio spectra was manipulated, namely; ratio difference, ratio subtraction and mean centering. Ratio difference and mean centering methods have the advantage of complete elimination
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Table 4 Statistical comparison between the results obtained by the proposed spectrophotometric methods and reported methods [23] for the determination of PARA and DANT in pure powder form. Items
Mean RSD%a SEMg Variance n Student’s t-testd F value a b c d e f g
PARA
DANT
RD
RS/AR
MC
AS
AM
Reported method [23]b
RD
MC
AR
AS
AM
Reported method [23]c
99.77 0.67 0.25 0.4413 7 0.104 1.128e
100.46 0.50 0.19 0.2574 7 1.776 1.934e
99.77 0.69 0.26 0.4739 7 0.112 1.065e
100.00 0.66 0.25 0.4343 7 0.443 0.808e
99.99 0.88 0.33 0.7787 7 0.328 1.565f
99.82 0.71 0.35 0.4977 4
99.46 0.77 0.29 0.5857 7 0.760 1.021f
99.45 0.78 0.29 0.6017 7 0.788 1.053f
100.39 0.64 0.24 0.4124 7 1.309 1.391e
99.90 0.67 0.25 0.4502 7 0.156 1.274e
99.98 0.54 0.20 0.2898 7 0.382 1.979e
99.83 0.76 0.38 0.5735 4
Percent relative standard deviation. Reported method for PARA is based on first derivative spectrophotometric method by measuring the peaks amplitudes at 265.5 nm. Reported method for DANT is based on zero order spectrophotometric method by measuring the absorbance at 380 nm. The corresponding tabulated value of t equals to 2.2621at p = 0.05. The corresponding tabulated value of F equals to 4.7570 at p = 0.05. The corresponding tabulated value of F equals to 8.9408 at p = 0.05. SEM is the standard error of the mean.
Table 5 One way ANOVA testing for the different proposed and reported methods used for the determination of PARA and DANT. Source of variation
DF
Sum of squares
Mean square
F value
PARA
Between experiment Within experiment
5 33
2.362 15.767
0.472 0.478
0.989 (2.503)
DANT
Between experiment Within experiment
5 33
3.171 14.952
0.634 0.453
1.399 (2.503)
The values between parentheses are the theoretical F values. The population means are not significantly different.
of interfering component in the form of a constant so no need for critical measurements which lead to reproducible and robust results. Ratio subtraction has the ability of obtaining spectra typical to that of the pure form which confirms the spectral profile of each component of interest. But it determines the non-extended drug only. However, those methods require the presence of a standard solution of the interfering substance to act as a divisor in addition to the proper choice of the divisor which has a critical effect on signal to noise ratio. All developed methods do not involve several steps to analyze each drug alone, or other sophisticated calculation. As a final conclusion, the proposed methods could be easily applied in quality control laboratories without any preliminary separation step as they showed good accuracy and precision as indicated from the obtained result. References [1] S.C. Sweetman, Martindale, The Complete Drug Reference, 33rd ed., Pharmaceutical Press, London, 2002. [2] V. Maslarska, J. Tenchevam, Int. J. Pharm. Pharm. Sci. 5 (2013) 417–419. [3] British Pharmacopoeia, Her Majestyis Stationery Office, London, 2009. [4] European Pharmacopoeia, 6th ed., 2007. [5] A.D. Likhar, K.R. Gupta, S.G. Wadodkar, Int. J. Pharm. Pharm. Sci. 2 (2010) 156– 161. [6] T.T.A. Duong, D.V. Hoang, Asian J. Res. Chem. 2 (2009) 143–147. [7] S.J. Babar, V.B. Mane, S.B. Bhise, Int. J. Pharm. Pharm. Sci. 4 (2012) 206–209. [8] S.S. Zarapkar, U.P. Hulkar, N.P. Bhandari, Indian Drugs 36 (1999) 710–713. [9] B. Tsvetkova, I. Pencheva, P. Peikov, Int. J. Biol. Pharm. Allied Sci. 1 (2012) 913– 917. [10] Y.R. Liang, J. Hu, H.L. Wu, L.Y. Le, W.J. Wang, Yaowu Fenxi Zazhi 26 (2006) 411– 414. [11] A.P. Argekar, J.G. Sawant, J. Planar Chromatogr. Mod. TLC 12 (1999) 361–364. [12] E. Deconinck, P.Y. Sacre, S. Baudewyns, P. Courselle, J. Beer, J. Pharm. Biomed. Anal. 56 (2011) 200–209. [13] M. Godejohann, L.H. Tseng, U. Braumann, J. Fucher, M. Spraul, J. Chromatogr. A 1058 (2004) 191–196.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A comparative study of smart spectrophotometric methods for simultaneous determination of a skeletal muscle relaxant and an analgesic in combined dosage form Hesham Salem c, Dalia Mohamed a,b,⇑ a b c
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, 11787 6 October City, Egypt Analytical Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, 11795 Cairo, Egypt Analytical Chemistry Department, Faculty of Pharmacy, Deraya University, 14511 Cairo, Egypt
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Six different spectrophotometric
methods based on isosbestic point or ratio spectra. All methods could be applied for simultaneous analysis of PARA and DANT. All methods could be easily applied in QC labs. No significant difference in accuracy and precision compared to reported method. They are applied for analysis of the dosage form without interference of excipients.
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 5 December 2014 Accepted 28 December 2014 Available online 3 January 2015 Keywords: Absorbance subtraction method Amplitude modulation method Dantrelax compoundÒ capsules Dantrolene sodium Paracetamol
a b s t r a c t Six simple, specific, accurate and precise spectrophotometric methods were developed and validated for the simultaneous determination of the analgesic drug; paracetamol (PARA) and the skeletal muscle relaxant; dantrolene sodium (DANT). Three methods are manipulating ratio spectra namely; ratio difference (RD), ratio subtraction (RS) and mean centering (MC). The other three methods are utilizing the isoabsorptive point either at zero order namely; absorbance ratio (AR) and absorbance subtraction (AS) or at ratio spectrum namely; amplitude modulation (AM). The proposed spectrophotometric procedures do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined. The selectivity of the developed methods was investigated by analyzing laboratory prepared mixtures of the drugs and their combined dosage form. Standard deviation values are less than 1.5 in the assay of raw materials and capsules. The obtained results were statistically compared with each other and with those of reported spectrophotometric ones. The comparison showed that there is no significant difference between the proposed methods and the reported methods regarding both accuracy and precision. Ó 2015 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author at: Analytical Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, 11795 Cairo, Egypt. Mobile: +20 1000664243. E-mail address: [email protected] (D. Mohamed). http://dx.doi.org/10.1016/j.saa.2014.12.099 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Paracetamol (PARA); N-acetyl-p-aminophenol; (Fig. 1a) is a widely used analgesic and antipyretic agent for the relief of fever, headaches and minor pains. It is a major ingredient in numerous cold and flu remedies. In combination with non-steroidal anti-
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inflammatory drugs and opioid analgesics; paracetamol is also used in the management of severe pain (such as post-operative pain) [1,2]. Paracetamol alone or in combination with other drugs was estimated by titrimetry [3,4], spectrophotometric method [5– 7], HPLC [8,9], TLC [10], HPTLC [11], UHPLC [12], LC–MS [13], FT-IR [14], amperometric determination [15] and fluorimetry [16]. Dantrolene sodium (DANT); 1-[5-(4-nitrophenyl)furfurylidene amino]imidazolidine-2,4-dione sodium salt (Fig. 1b) is a muscle relaxant with a direct action on skeletal muscles [1]. DANT uncouples muscular contraction from excitation, probably by interfering with the release of calcium from the sarcoplasmic reticulum [1]. DANT was also determined either alone, in presence of its metabolites and impurities or in biological fluids utilizing different methods as HPLC [17–20] and polarography [21]. Most of the drugs used for treatment of skeletal muscle disorders are combined with analgesics such as PARA. PARA and DANT are formulated as capsule where this combination is used for myalgia, sprains and skeletal muscle spasms due to neurological disorder. Literature search revealed only two methods for the simultaneous determination of PARA and DANT in dosage form; HPLC-UV [22] and spectrophotometry based on zero order, first derivative and derivative ratio methods [23]. The main problem of spectrophotometric binary or ternary mixture analysis is the simultaneous determination of the compounds in the same mixture without prior separation. Several spectrophotometric methods have been used for resolving such mixtures with overlapped spectra such as; solving two simultaneous equations (Vierodet’s method) [24], Derivative spectrophotometry [25], ratio derivative spectrophotometry [26,27], in addition to, H-point standard addition method [28]. Thus, the aim of this work was to develop and conduct comparative study on recently developed well established spectrophotometric methods based either on ratio spectra (RD, RS, MC) or on isosbestic point (AR, AS and AM) for resolving binary mixture of spectral interfering problem without preliminary separation. The utilized methods are very simple, accurate, precise and do not require any sophisticated apparatus. Theory Absorbance ratio method (AR) Erk [29] developed a spectrophotometric method which is based on the linear relationship between the absorbance ratio value of a binary mixture of X and Y and the relative concentration of such a mixture. The main criteria for the application of this method are the existence of an isoabsorptive point and the selection of optimum wavelengths (k1, k2, kiso) which can fulfill good determination of the components in the binary mixture. Absorbance subtraction method (AS) This method is based on the same principles as the absorbance factor method [30–33]. However, this method could be applied for
167
the analysis of a mixture of two drugs X and Y having overlapped spectra intersecting at isoabsorptive point where Y is extended over X, and X doesn’t show any contribution at another wavelength (k2). In this method, the isoabsorptive point kiso could be used for separate quantitative estimation of each X and Y in their mixture (X + Y). The determination can be done using mathematically calculated factor of one of these components. By simple manipulation step, we can obtain the absorbance value corresponding to X and Y, separately. So, the concentration of each component could be obtained via the isoabsorptive point regression equation without any need for a complementary method. Amplitude modulation method (AM) The method is a ratio spectrum manipulating method using the normalized spectrum of the divisor obtained by dividing certain spectrum of Y0 component by its concentration [30,32]. For a mixture of X and Y, where Y is more extended than X; X and Y shows isoabsorptive point at the zero order spectrum and consequently is retained as an isosbestic point at the ratio spectrum. Experimental Apparatus and software A JASCO V-530 double beam UV–VIS spectrophotometer loaded with Spectra Manager Program (JASCO) was used for spectral acquisition and elaboration of the data obtained. Quartz cuvettes, 1-cm pathlength were used for measuring the light absorption in the ultraviolet region (200–400 nm). For the mean centering for ratio spectra method, MatlabÒ Version 7.9 was used. Chemicals Pure samples: paracetamol (PARA) was kindly supplied by International Drug Agency for Pharmaceutical Industry (Portsaid, Egypt). Dantrolene sodium (DANT) was kindly supplied by Chemipharm Pharmaceutical Industries (6th October City, Egypt). Their purity was found to be 99.82 ± 0.71 and 99.83 ± 0.76 for PARA and DANT, respectively, by a reported method [23]. Market sample: Dantrelax compoundÒ capsules labeled to contain 300 mg PARA and 25 mg DANT (Batch number: 131186A), was manufactured by Chemipharm Pharmaceutical Industries (6th October City, Egypt). Solvents: methanol was obtained from Merck (Darmstadt, Germany). Standard solutions Separate stock solutions of PARA and DANT (1 mg/mL) were prepared by dissolving the compounds in methanol then completing in 100 mL measuring flasks. The working solutions were freshly prepared by dilution from the stock solutions with the same solvent to obtain a concentration of (0.1 mg/mL) for each of PARA and DANT. Procedures Spectral characteristics The zero-order absorption spectra (D0) of the two components were recorded at (200–400 nm) against methanol as a blank.
Fig. 1. Chemical structures of (a) paracetamol (PARA), (b) dantrolene sodium (DANT).
Construction of calibration curves Ratio difference method (RD). Standard solutions containing 1–30 lg/mL PARA and 1–40 lg/mL DANT were prepared separately in methanol. The absorption spectra of the resulting
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solutions were measured and divided by the absorption spectra of 25 lg/mL DANT and 20 lg/mL PARA, where the obtained ratio spectra were recorded. Construct calibration curves for PARA and DANT by plotting the difference between the amplitudes of the obtained ratio spectra at 253 and 218 nm and 218 and 253 nm respectively, versus the corresponding concentrations. The regression equations were computed. Ratio subtraction method (RS). Standard solutions of PARA containing 1–30 lg/mL were prepared in methanol. The absorption spectra of the resulting solutions were measured. Construct calibration curves relating the zero order spectra of PARA at 248 nm versus the corresponding concentrations and compute the regression equations. Mean centering method (MC). Standard solutions containing 1– 30 lg/mL PARA and 1–40 lg/mL DANT were prepared separately in methanol. The absorption spectra of the resulting solutions were measured and divided by the absorption spectra of 25 lg/mL DANT and 20 lg/mL PARA, where the obtained ratio spectra were recorded and then mean centered. Construct calibration curves relating the amplitudes of the mean centered values to the corresponding concentrations at 253 nm for PARA and 218 nm for DANT. Absorbance ratio method (AR). Separate standard solutions of PARA and DANT each containing 1–30 lg/mL were prepared separately in methanol. The absorption spectra of the resulting solutions were measured. Construct calibration curves relating the zero order spectra of PARA at 248 nm and DANT at 380 nm versus the corresponding concentrations and compute the regression equations. Absorbance subtraction method (AS). Separate standard solutions of PARA and DANT each containing 1–30 lg/mL were prepared separately in methanol. The absorption spectra of the resulting solutions were measured. Construct calibration curves relating the zero order spectra of PARA or DANT at 274 nm versus the corresponding concentrations and compute the regression equations. The absorbance factor of DANT [A274/A380] was calculated and was found to be 0.27. Amplitude modulation method (AM). Separate standard solutions of PARA and DANT each containing 1–30 lg/mL were prepared separately in methanol. The absorption spectra of the resulting solutions were measured and divided by the normalized divisor of DANT spectrum, and the obtained ratio spectra were recorded. The calibration curves were constructed by plotting the amplitudes of PARA or DANT at 274 nm against their corresponding concentrations and the regression equations were computed. Application to laboratory prepared mixtures Into a series of 10-mL volumetric flask, accurate aliquots of PARA and DANT were transferred from their working solutions to prepare seven mixtures containing different ratios of the cited drugs. The volumes were completed with methanol. The spectra of the prepared solutions were recorded at (200–400 nm). The concentration of each drug was calculated by substitution in the corresponding regression equation after applying the corresponding manipulating steps for each method. In case of ratio subtraction method, the mixtures were divided by 25 lg/mL DANT as a divisor followed by subtraction of the obtained constant from the division at the plateau (310–380 nm), then multiply the obtained spectrum by the spectrum of 25 lg/mL of DANT. While in case of absorbance ratio method; the analysis of PARA and DANT in binary mixture was performed by using the following equations:
C 1 ¼ ðQ 1 b1 Þ=a1 ðAiso =aiso Þ C 2 ¼ ðQ 2 b2 Þ=a2 ðAiso =aiso Þ where Q1 = A1/Aiso for PARA, Q2 = A2/Aiso DANT. C1, C2 = concentrations of PARA and DANT, respectively. Aiso = absorbance at isoabsorptive point, where kiso = 274 nm. aiso = absorptivity at isoabsorptive point = Aiso/C1 + C2, a1 = slope of regression equation (Q1 versus C1/C1 + C2), a2 = slope of regression equation (Q2 versus C2/C1 + C2), b1, b2 = intercept values for these regression equations. A1 and A2 denote the absorbances of the mixture solution measured at k1 (248 nm) and k2 (380 nm). Application to pharmaceutical preparation Ten capsules were evacuated and the content was well mixed. An accurate amount of the powder equivalent to 120 mg PARA and 10 mg DANT was weighed and transferred into a beaker, 50 mL methanol were added with continuous stirring for about 10 min. The solution was filtered into 100-mL volumetric flask. The volume was completed with methanol. Aliquots equivalent to 0.15 mL from this solution were transferred to 10-mL volumetric flasks and the volume was completed with methanol to obtain a final concentration of 18 lg/mL for PARA and 1.5 lg/mL for DANT. The concentration of each drug was calculated using the corresponding regression equation. When carrying out the standard addition technique, different known concentrations of pure standard of each drug were added to the pharmaceutical dosage form before proceeding in the previously mentioned methods. Results and discussion The use of spectrophotometric methods such as derivative spectrophotometry, ratio spectra spectrophotometry and other chemometric spectral calibration techniques is crucial for analytical studies, related to the quality control and routine analysis of commercial products in the research or industry laboratories. It was found that these spectrophotometric methods are preferable instead of hyphenated analytical instrumentations or techniques such as LC–MS, GC–MS and LC–NMR which always require prior extraction and separation steps and other tedious analytical process during analysis. On the other hand, the related techniques having complex components bring high cost and time consumption. Taking into account all above arguments, the quantitative spectrophotometric resolution of the mixtures of two or more compounds having overlapped spectra is an interesting issue for the analytical chemistry. Besides, the existing spectrophotometric methods were found to be very easy to apply, very rapid, sensitive and yet very cheap for analysis of mixture. Fig. 2 shows the overlapping zero-order absorption spectra (D0) of PARA and DANT. Although DANT can be determined at its kmax at 380 nm, however, this does not provide simultaneous determination of both drugs. In addition DANT shows high absorbance at 248 nm hindering the determination of PARA at its kmax (248 nm). Thus, the main task of this work was to establish simple, sensitive and accurate spectrophotometric methods for the simultaneous determination of paracetamol and dantrolene sodium in their bulk powders and pharmaceutical dosage form with satisfactory precision for good analytical practice. In addition, to construct a statistical comparison between the ability of the proposed methods in determining the two drugs. Ratio difference method (RD) The overlapped spectra of PARA and DANT suggested that ratio difference spectrophotometric method is as a suitable method for their simultaneous determination. Lotfy and Hegazy [34]
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Fig. 2. Absorption spectra of 20 lg/mL PARA (_____), 20 lg/mL DANT (— – — –) and a binary mixture of 10 lg/mL of each PARA and DANT (– – – –).
introduced the ratio difference spectrophotometric method (RD) which depends on measuring the difference between two amplitude values at two wavelengths in one step using the ratio spectra, so it eliminates the derivative steps and therefore the signal to noise ratio is enhanced [35–37]. The linearity of the amplitude values at each of the chosen wavelength against the corresponding concentration should be checked to ensure minimal noise to signal ratio at these wavelengths. By applying the ratio difference method, the difference between the amplitude values is measured by subtraction the two values at two selected wavelength, so the noise will be cancelled. For the determination of PARA concentration by this method, the only requirement was the contribution of PARA and DANT at the two selected wavelengths (253 and 218 nm) where the ratio spectrum of DANT showed the same amplitudes (constant) whereas PARA showed significant difference in these two amplitude values at these two selected wavelengths with concentration. Similarly, DANT could be estimated at the selected wavelengths (218 and 253 nm). PARA (1–30 lg/mL) spectra were divided by the spectrum of DANT (25 lg/mL), Fig. 3a, and DANT (1–40 lg/mL) spectra were divided by the spectrum of PARA (20 lg/mL), Fig. 3b. The concentration of PARA was calculated by using the regression equation representing the linear relationship between the differences of these ratio spectra amplitudes at the two selected wavelengths versus the corresponding concentration of the drug. Similarly, DANT could be determined in the same manner. Different concentrations of PARA (10, 20 and 25 lg/mL) and of DANT (15, 25 and 35 lg/mL) were tested as a divisor but the concentrations 20 lg/mL of PARA and 25 lg/mL of DANT gave minimum noise, smoother ratio spectra and maximum sensitivity. The selected wavelengths were the best regarding average recovery percent when used for the prediction of PARA and DANT concentrations in bulk powder as well as in laboratory prepared mixtures. Ratio subtraction method (RS) The ratio subtraction method (RS) [38] was used for the determination of PARA concentration in the binary mixture of PARA and DANT. The mixture’s spectrum is divided by using certain concentration of one component as a divisor, where the constant value is calculated along the plateau region which is parallel to the baseline, then subtracted from the ratio spectra followed by multiplication by the divisor. In ratio subtraction the component of interest is recovered in its zero order spectra and measured at its kmax to minimize the noise error.
Fig. 3. Ratio spectra of: (a) PARA (1–30 lg/mL) and 25 lg/mL DANT (- - - -) using 25 lg/mL DANT as a divisor and (b) DANT (1–40 lg/mL) and 20 lg/mL PARA (- - - -) using 20 lg/mL PARA as a divisor, both spectra showing the two selected wavelengths (218 and 253 nm).
Thus, PARA can be determined by dividing the spectrum of the binary mixture by a known concentration of DANT as a divisor (25 lg/mL). The constant obtained from the division was measured at the plateau (310–380 nm). By subtracting this constant value, then multiply the obtained curve after subtraction by the spectrum of 25 lg/mL of DANT (the divisor), therefore we can recover the zero order absorption spectrum of PARA present in the mixture, Fig. 4, and measured at its kmax (248 nm). Mean centering method (MC) The obtained absorption spectra of the mixture are divided by the spectrum of standard solution of one component and then mean centered (Fig. 5). PARA has been determined at 253 nm in a concentration range of 1–30 lg/mL, using the spectrum of DANT (25 lg/mL) as a divisor, while, DANT was determined at 218 nm in a concentration range 1–40 lg/mL, using the spectrum of PARA (20 lg/mL) as a divisor. The careful choice of the divisor and the working wavelengths were of great importance, consequently different concentrations of PARA (10, 20 and 25 lg/mL) and of DANT (15, 25 and 35 lg/mL) were tried as divisors. It was found that concentrations 20 lg/mL of PARA and 25 lg/mL of DANT gave minimum noise and better selectivity. Absorbance ratio method (AR) The absorbance ratio method was applied to determine the concentration of PARA (C1) and DANT (C2) in a binary mixture through the linear relationship between the absorbance ratio value of the binary mixture of PARA/DANT and the relative concentration in that mixture. The main criteria for the application of the absorbance ratio method are the existence of an isoabsorpative point and selection of optimum wavelengths. For good separation, the
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Fig. 5. Mean centered ratio spectra of: (a) PARA (1–30 lg/mL) using 25 lg/mL DANT as a divisor and (b) DANT (1–40 lg/mL) using 20 lg/mL PARA as a divisor.
Fig. 4. (a) Division spectra of laboratory prepared mixtures using 25 lg/mL DANT as a divisor, (b) division spectra after subtraction of the constants, (c) the obtained zero order spectra of PARA after subtraction of constant and multiplication by the spectrum of 25 lg/mL DANT.
absorbance of PARA was obtained by subtraction. The absorbance values of PARA and DANT at kiso 274 nm were used to calculate each of their concentration using the unified regression equation at the same wavelength.
ADANT at 274 nm ¼ ½A274 =A380 : ðAmix at 380 nmÞ APARA at 274 nm ¼ Amix at 274 nm ADANT at 274 nm:
method should be applied using the wavelengths (k1, k2, kiso) which can fulfill good determination of the components in the binary mixture [29]. The choice of the two wavelengths (248 and 380 nm) beside the isoabsorpative point (274 nm) gave the best recovery percentages for determination of laboratory prepared mixtures, (Fig. 2). The absorbance of the previous laboratory prepared mixtures at kiso (274 nm) were plotted against total concentrations of (C1) and (C2) and the regression equation was computed, to obtain slope of the regression line (aiso = 0.0174). The absorbance of each solution was measured at k1 (248 nm), k2 (380 nm) and kiso (274 nm), the relative absorbance (Q1 = A1/Aiso) were plotted against the relative concentration (C1/C1 + C2) and the regression equation parameters were computed (b1/a1 = 0.257). The relative absorbance (Q2 = A2/Aiso) were plotted against the relative concentration (C2/C1 + C2) and the regression equation parameters were computed (b2/a2 = 0.0041).
Absorbance subtraction method (AS) This method is based on the isosbestic point present in zero order absorption spectra known as the isoabsorptive point, where the components exhibiting this point have equal absorptivities. For the determination of PARA and DANT, their isosbestic point at 274 nm was utilized, Fig. 2. The absorbance of DANT at 274 nm was calculated using its absorbance factor at this point (where [A274/A380] for DANT was found to be 0.27), and then the
where Amix is the absorbance of the binary mixture, ADANT and APARA are the absorbance of DANT and PARA, respectively; and [A274/A380] is the absorbance factor of pure DANT at 274 nm to that at 380 nm. The requirements of this method are the existence of isoabsorptive point of both components and the extension of the spectra of one component. The advantage of the absorbance subtraction method over the conventional isoabsorpative point is that there is no need for a complementary spectrophotometric method to measure the concentration of one of the components. Both components can be determined using unified regression equation at kiso. The disadvantage of this method is the increased risk of error in calculating the absorbance factor in case of low concentrations of the extended component (DANT) or its low value of absorbance at extension region. Amplitude modulation method (AM) This proposed method is based on two facts; foremost the isosbestic point whenever present in an absorption spectrum will be retained at the same point even after division by one component as a divisor in the ratio spectrum. As known the results of manipulating ratio spectra techniques are greatly affected by the choice of the divisor. Thus, to eliminate the effect of the divisor, the normalized spectrum of DANT was used. By dividing the spectrum of the binary mixture by the normalized DANT divisor spectrum, we obtained the ratio spectra, Fig. 6. The amplitude value of the constant was determined at the plateau region at (310–380 nm), which was equal to the amplitude of the
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Linearity and range The linearity of the proposed methods was evaluated by processing the different calibration curves on three different days. The calibration curves were constructed within concentration ranges that were selected on the basis of the anticipated drugs concentration during the assay of the dosage form. The linearity of the calibration curves were validated by the high value of correlation coefficients. The analytical data of the calibration curve including concentration ranges and calibration equations are summarized in Table 1. Accuracy
Fig. 6. Ratio spectra of a mixture of 20 lg/mL of each PARA and DANT using normalized DANT divisor after subtraction of the constant.
constant of DANT along the whole spectrum. At the isosbestic point (kiso) at 274 nm, the amplitude of the ratio spectra at this point was equal to the sum of the amplitudes of DANT and PARA. By applying ratio manipulation using the normalized divisor, the curve of interfering substance (DANT) was converted to a constant, which is a straight line parallel to wavelength axis having the same amplitude, so there was no need for calculation of response factor. By subtracting the previously obtained constant, we obtained the corresponding amplitude of PARA, which was equivalent to recorded concentration of PARA in the mixture (CRecorded of PARA). To eliminate any error due to signal to noise ratio, the actual concentration of DANT or PARA were calculated by using their corresponding unified regression equation at kiso 274 nm.
C Recorded ¼ 0:9999C þ 0:0186: where CRecorded represents the recorded amplitude of ratio spectrum which was equal to the recorded concentration of PARA and C represented corresponding concentration of PARA. The requirements of this method are the existence of isoabsorptive point of both components at zero order spectrum and consequently in the ratio spectra; and the extension of the spectra of one component. The advantage of amplitude modulation method over other mathematical techniques utilizing the constant is the reduced manipulation steps and only one divisor is needed in order to determine both components in the mixture. By using the normalized divisor, the results are not affected by the choice of divisor and it minimizes the noise in the obtained ratio spectra, where the recorded amplitudes are directly modulated to concentrations in order to minimize the calculation steps. This divisor was obtained by dividing certain spectrum of DANT by its concentration or by dividing the addition of several spectrum of DANT by the sum of their corresponding concentrations. This method has advantage over the isoabsorpative point at zero order that it measures the concentration of both components with no need for other conventional method to measure one of the components in the mixture. This method has advantages over the newly developed absorbance subtraction method (AS) through using the normalized divisor, where the obtained amplitude at the ratio spectrum will directly represent the concentration of each component and so the risk of error upon the determination of absorbance factor of lower absorbance will be reduced through the elimination of the absorbance factor step. Methods validation Methods validation was performed according to ICH guidelines [39] for the proposed methods as follows:
To study the accuracy of the proposed methods, procedures under construction of the calibration curves, for both drugs using the proposed methods, were repeated three times for the determination of seven different concentrations of pure PARA and DANT within the linearity range. The accuracy expressed as percentage recoveries (mean) and standard deviation was shown in Table 1. Good accuracy of the developed methods was indicated by the results obtained. Precision The intra-day and inter-day precision of the proposed methods were determined by the analysis of three different concentrations of pure PARA and DANT, within the linearity range, by three replicate analyses of three pure samples of both drugs on a single day and on three consecutive days, respectively. The results were illustrated in Table 1. Selectivity Selectivity was ascertained by analyzing different mixtures containing both drugs in different ratios within the linearity range. Satisfactory results were shown in Table 2. Good recovery percentages with accepted standard deviations were obtained in all cases. Limits of detection and quantification The LOD is the lowest concentration of the drug that can be detected, but not necessarily quantitated, under the stated experimental conditions, while, the LOQ is the lowest concentration of the analyte that, can be determined with acceptable precision and accuracy. Both LOD and LOQ were calculated and the results are abridged in Table 1. Application of the proposed methods The proposed procedures were applied for the determination of PARA and DANT in Dantrelax compoundÒ capsules where the obtained recovery and standard deviation proved no excipients’ interference. The validity of the proposed procedures is further assessed by applying the standard addition technique. The results obtained are shown in Table 3. Statistical analysis Table 4 showed statistical comparisons of the results obtained by the proposed methods and reported method [23]. The calculated t and F values were less than the theoretical ones indicating that there was no significant difference between the proposed and the reported methods with respect to accuracy and precision. Oneway ANOVA was applied for the purpose of comparison of devel-
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Table 1 Assay parameters and validation sheet obtained by applying the proposed spectrophotometric methods. Parameter
RD
Wavelength (nm) Calibration rangea (lg/mL) Slope Intercept Correlation coefficient Mean RSD% LOD LOQ Repeatabilityab Inter-day precisionab a b
RS
MC
AR
AS
AM
PARA
DANT
PARA
PARA
DANT
PARA
DANT
PARA
P1–P2 253–218 1–30 0.1669 0.0165 0.9997 99.77 0.67 0.19 0.58 0.61 0.75
P1–P2 218–253 1–40 0.0556 0.0414 0.9997 99.46 0.77 0.18 0.55 0.59 0.79
D0 at 248 1–30 0.0857 0.005 0.9998 100.46 0.50 0.21 0.64 0.65 0.71
MC at 253 1–30 0.122 0.0159 0.9996 99.77 0.69 0.28 0.85 0.71 0.85
MC at 218 1–40 0.0391 0.0413 0.9997 99.45 0.78 0.23 0.70 0.62 0.65
D0 at 248 1–30 0.0857 0.005 0.9998 100.46 0.50 0.21 0.64 0.65 0.71
D0 at 380 1–30 0.0655 0.0108 0.9997 100.39 0.64 0.23 0.70 0.69 0.84
D0 at 274 1–30 0.0174 0.0040 0.9999 100.00 99.90 0.66 0.67 0.18 0.55 0.74 0.65 0.79 0.82
DANT
PARA
DANT
P at 274 1–30 0.9999 0.0186 1.000 99.99 99.98 0.88 0.54 0.30 0.91 0.77 0.65 0.94 0.81
Average of three experiments. Relative standard deviation of three concentrations of each drug (5, 15, 25 lg/mL).
Table 2 Analysis of laboratory prepared mixtures by the proposed spectrophotometric methods. Conc in lg/mL PARA:DANT
10:20 5:25 12:1b 10:10 20:10 25:5 24:2b Mean RSD%a SEMc Variance a b c
RS
MC
PARA
RD DANT
PARA
PARA
DANT
AR PARA
DANT
AS PARA
DANT
AM PARA
DANT
98.64 98.22 98.09 100.40 99.12 98.25 100.19 98.99 0.97 0.37 0.9227
101.42 103.05 100.29 101.35 103.44 102.25 101.50 101.90 1.07 0.40 1.1838
99.79 100.55 100.35 98.63 98.76 98.92 99.27 99.47 0.78 0.29 0.5970
99.90 99.21 99.65 100.22 98.63 98.30 99.96 99.41 0.73 0.27 0.6557
99.79 100.71 100.45 100.36 100.63 100.53 98.94 100.20 0.63 0.24 0.6087
101.27 99.26 100.05 98.82 101.16 100.80 99.46 100.12 0.97 0.37 0.9514
100.30 98.72 100.04 100.60 98.76 99.17 98.71 99.47 0.82 0.31 0.6697
99.90 99.50 100.72 100.15 98.51 98.91 99.46 99.59 0.75 0.28 0.5567
99.12 100.99 99.35 100.12 99.95 99.12 98.38 99.58 0.85 0.32 0.7225
97.60 98.40 99.83 98.70 98.60 98.24 99.20 98.65 0.72 0.27 0.5068
100.88 100.02 102.00 100.02 99.99 101.20 101.40 100.79 0.79 0.30 0.6400
Percent relative standard deviation. Ratio present in dosage form. SEM is the standard error of the mean.
Table 3 Application of standard addition technique to the analysis of dosage form by applying the proposed spectrophotometric methods. Methods
RD RS MC AR AS AM a b c
PARA
DANT
Found in lg/mLa
b
17.76 18.14 17.90 17.91 18.00 17.91
98.69 ± 0.64 100.77 ± 1.00 99.46 ± 0.77 99.52 ± 0.75 100.00 ± 0.59 99.50 ± 1.07
Recovery % ± SD
Pure addedc recovery % ± SD
Found in lg/mLa
b
Pure addedc recovery % ± SD
98.44 ± 1.05 100.82 ± 1.07 99.36 ± 0.84 99.37 ± 0.92 99.87 ± 0.77 100.13 ± 0.91
1.52
101.60 ± 1.10
101.25 ± 1.03
1.51 1.50 1.49 1.51
100.51 ± 0.76 100.00 ± 0.87 99.33 ± 0.87 100.53 ± 0.86
100.10 ± 0.89 99.35 ± 0.54 99.61 ± 0.96 99.92 ± 0.72
Recovery % ± SD
PARA claimed to be 18 lg/mL, DANT claimed to be 1.5 lg/mL. Average of five experiments. Average of three experiments (added equivalent to 2, 4, 6, 8, 12 lg/mL PARA and 0.5, 1, 3.5, 8.5, 20.5 lg/mL DANT).
oped methods; where Table 5 shows no significant difference between the developed methods. Conclusion This work describes six simple, specific, accurate and precise spectrophotometric methods for the simultaneous determination of paracetamol and dantrolene sodium in bulk powder, in laboratory prepared mixture and in dosage form. Three of the performed methods namely; absorbance ratio, absorbance subtraction and amplitude modulation have the ability to analyze the two drugs simultaneously and all require the presence of isoabsorptive point. Absorbance ratio has the advantage that spectral measurements are done at zero order which allows maximum accuracy and reproducibility but unfortunately it requires multiple calculation steps.
Absorbance subtraction allows the determination of the two interfering components using unified regression equation at kiso. However, the disadvantage of this method is the increased risk of error in calculating the absorbance factor in case of low concentrations of the extended component. Amplitude modulation has advantages over the newly developed absorbance subtraction method through using the normalized divisor, where the obtained amplitude at the ratio spectrum will directly represent the concentration of each component and so the risk of error upon the determination of absorbance factor of lower absorbance will be reduced through the elimination of the absorbance factor step. In addition to the previous methods, three other methods were performed in which ratio spectra was manipulated, namely; ratio difference, ratio subtraction and mean centering. Ratio difference and mean centering methods have the advantage of complete elimination
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Table 4 Statistical comparison between the results obtained by the proposed spectrophotometric methods and reported methods [23] for the determination of PARA and DANT in pure powder form. Items
Mean RSD%a SEMg Variance n Student’s t-testd F value a b c d e f g
PARA
DANT
RD
RS/AR
MC
AS
AM
Reported method [23]b
RD
MC
AR
AS
AM
Reported method [23]c
99.77 0.67 0.25 0.4413 7 0.104 1.128e
100.46 0.50 0.19 0.2574 7 1.776 1.934e
99.77 0.69 0.26 0.4739 7 0.112 1.065e
100.00 0.66 0.25 0.4343 7 0.443 0.808e
99.99 0.88 0.33 0.7787 7 0.328 1.565f
99.82 0.71 0.35 0.4977 4
99.46 0.77 0.29 0.5857 7 0.760 1.021f
99.45 0.78 0.29 0.6017 7 0.788 1.053f
100.39 0.64 0.24 0.4124 7 1.309 1.391e
99.90 0.67 0.25 0.4502 7 0.156 1.274e
99.98 0.54 0.20 0.2898 7 0.382 1.979e
99.83 0.76 0.38 0.5735 4
Percent relative standard deviation. Reported method for PARA is based on first derivative spectrophotometric method by measuring the peaks amplitudes at 265.5 nm. Reported method for DANT is based on zero order spectrophotometric method by measuring the absorbance at 380 nm. The corresponding tabulated value of t equals to 2.2621at p = 0.05. The corresponding tabulated value of F equals to 4.7570 at p = 0.05. The corresponding tabulated value of F equals to 8.9408 at p = 0.05. SEM is the standard error of the mean.
Table 5 One way ANOVA testing for the different proposed and reported methods used for the determination of PARA and DANT. Source of variation
DF
Sum of squares
Mean square
F value
PARA
Between experiment Within experiment
5 33
2.362 15.767
0.472 0.478
0.989 (2.503)
DANT
Between experiment Within experiment
5 33
3.171 14.952
0.634 0.453
1.399 (2.503)
The values between parentheses are the theoretical F values. The population means are not significantly different.
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