A Review on Methods Developed for Estimation of Paracetamol in Combination with Other Drugs
Satyam Baghel^{1}, Kamal Shah^{1*}
^{1}Institute of Pharmaceutical Research, GLA University, Mathura, UP, India 281406.
*Email: [email protected]
ABSTRACT
In this evergrowing world, it is crucial to improve upon the formulations in terms of potency, patient acceptability, fewer side effects, and quicker relief. Due to these requirements, the market is flooded with various combination dosage forms, with a constant increase in number. Paracetamol is a commonly used nonsteroidal antiinflammatory drug (NSAID) that has antipyretic and analgesic action. This drug is available in a wide range of combinations. It acts by inhibiting the production of prostaglandins, which combat pain and inflammation. A simultaneous multicomponent analysis is used to determine the estimation of medicines that are available in combination. Different analytical techniques are available for their determination, one of which includes the use of UV spectrophotometric methods. This review focuses on a variety of paracetamol combinations with drugs like Domperidone, Aceclofenac, Diclofenac Sodium, Etodolac, Ibuprofen, Piroxicam, Caffeine, Aspirin, and their simultaneous estimation by different UV methods viz. Simultaneous equation method, Absorbance ratio (QAnalysis), Difference spectrophotometry, Derivative spectroscopy method, and a few other chemometric methods. This manuscript would provide the platform to have exhaustive literature on methods used for the estimation of paracetamol with different drugs using a spectrophotometer. It would help the researchers and scholars who are working in the area.
Key words: Combination drugs, Multicomponent analysis, Paracetamol combinations, UV spectroscopy, Simultaneous
INTRODUCTION
Various drugs are prepared in various combinations and dosage forms because a large number of diseases that have harmful effects on humanity are universal. These multicomponent formulations are frequently favored because they have higher patient acceptance, enhanced efficacy, various actions, minimal side effects, and provide faster relief when handled appropriately [1]. Pharmaceutical formulations with a combination of drugs have shown promising benefits by counteracting other symptoms specific to a drug and formulation, and therefore the quantitative evaluation of such multicomponent formulations is critical.
One of the much more desired and extensively used equipment accessible for quantitative analysis is Absorption spectroscopy. The extent of light absorption is a result of an increase in the number and effectiveness of lightabsorbing molecules at a given wavelength [2]. The relation between the Concentration of the analyte and the quantity of light absorbed is the basis of the majority of analytical uses of molecular spectroscopy^{ }[3, 4]. BeerLambert Law states the same via the following expression –
(1) 
A is the absorbance of the compound at a given wavelength.
I_{0} is the Intensity of incident light on the cuvette.
I_{t} refers to the amount of light that is passed through the cuvette.
The molar concentration of the solute is represented by c.
l is the path length i.e., the distance traveled by the light inside the sample cell in cm.
Ɛ is the molar absorptivity. It is specific for every molecule undergoing electronic transition.
As a result of changes in the electronic energy of molecules or atoms brought on by energy absorption in the UV band (200–400 nm), electrons are excited from lower to higher energy levels (Figure 1). The amount of energy required for the transition of valence electrons in the molecule to happen is very precise and definite for the matter to be analysed^{ }[6].

Figure 1. Electronic and Vibrational Transitions 
These transitions are divided into two categories:
 Allowed transitions: Have an equal to or higher molar extinction coefficient (Ɛ_{MAX}) than 10^{4}. These are 
 σ ® σ*
 n ® σ*
 π ® π*
 Forbidden Transitions: These are the transitions for which the Ɛ_{MAX} value is lesser than 10^{4}.
 n ® π*
σ ® σ* transitions have the highest energy requirement, while n ® π* transitions have the least energy requirement [7].
Multicomponent analysis
One of the most sensitive and commonly used measurement techniques for quantitative and qualitative analysis is the simultaneous analysis of multiple components through absorbance measurements based on ultraviolet. This process avoids previous separation methods involving extraction, the concentration of components, and purification steps that make the process timeconsuming, and is fast, accurate, and simple; wide applicability to both organic and inorganic systems.
Simultaneous equation method
The concentration of different components with the additive nature of the absorbance present in the given mixture can be determined by solving a set of simultaneous equations even if their spectra overlap (Figure 2).
If a multicomponent system consists of two components M and N, each of which absorbs at λmax of the other, where λ_{1} is the wavelength of maximum absorbance of M (λ_{max} M) and λ_{2} is the Wavelength of maximum absorbance of N (λ_{max} N)
The information required is:
 a_{m1} and a_{m2} are the drug M’s absorptivity at λ_{1} and λ_{2} respectively.
 A_{n1} and a_{n2} are the drug N’s absorptivity at λ_{1} and λ_{2 }respectively.
 A_{1} and A_{2} represent the diluted sample's absorbance at wavelengths λ_{1 }and λ_{2} respectively.
C_{M} and C_{N} represent the concentrations of M and N in the sample, respectively.
At λ_{1},
(2) 
At λ_{2},
A_{2} = a_{M2 }b C_{M }+ a_{N2 }b C_{N} 
(3) 
If the cell is 1 cm, then b=1
C_{N} = (A_{1} a_{M2} A_{2} a_{M1})/ (a_{N1} a_{M2} – a_{N2} a_{M1}) 
(4) 
Similarly,
C_{X} = (A_{2} a_{N2} A_{1 }a_{N1})/ (a_{N1} a_{M2} – a_{N2} a_{M1}) 
(5) 
Using the abovementioned simultaneous equations, the drug concentrations of M and N in the combination may be simply computed.
Absorbance ratio method/Qanalysis
This approach is a variation of the Simultaneous equation technique. Its premise is based on the fact that given a chemical obeying Beer's Law, the absorbance ratios at any two wavelengths produce a constant value regardless of analyte concentration or path length^{ }[5]. A component at two distinct dilutions produces the same absorbance ratio of A_{1}/A_{2}. This is known as the k/a QValue ratio. In a twocomponent analysis, absorbance is measured at two wavelengths; one being the isosbestic point of the two substances (λ_{1}), the other being the wavelength of maximum absorption of any of the two components (λ_{2}) (Figure 2).
Two equations are constructed as in the previous method with a_{M1 }= a_{N2} at λ_{1 }and b = 1 cm;
A_{1 }= a_{M1}C_{M }+ a_{M1}C_{M} 
(6) 
A2A1=aM2CM+aM2CNaM1CM+aM1CN 
(7) 
The concentration of each component (C_{X} & C_{Y}) in the sample can be calculated
CM= QAQNAQQMQNaM1 
(8) 
CN= QAQMAQQMQNaN1 
(9) 
QN = Absorbance of sample solution at λ2 Absorbance of sample solution at λ1 
(10) 
QM = Absorptivity of pure component M at λ2 Absorptivity of pure component M at λ1 
(11) 
QN = Absorptivity of pure component N at λ2 Absorptivity of pure component N at λ1 
(12) 
A_{Q} = Absorbance of the sample at isosbestic (λ_{1}) wavelength
a_{M1} = Absorptivity of components M at isosbestic (λ_{1}) point
a_{N1} = Absorptivity of components N at isosbestic (λ_{1}) point
The precision of the dilutions of the sample solution and standard solution of M and N determines the accurate absorption and absorptivity measurements, respectively.
a) Vierordt’s Method 

b) Absorption Ratio Method 
Figure 2. Absorption spectra of substances M, N, and mixture 
Derivative spectrophotometry
Derivative spectroscopy is based on the principle of transition of simpler absorption spectrum into the first, second, or higher spectrum depending on their wavelength. This spectroscopic approach employs Gaussian bands to depict the modifying spectral data. It is also used for spectrum analysis to characterize any chemical configuration. The zeroth order spectrum, or fundamental absorption spectrum, is represented by the symbol D^{0 }[5]_{.}
Zeroorder spectra are simpler to understand than derivative spectra. The rate at which absorbance varies with wavelength is graphically depicted in a firstorder derivative spectrum. A firstorder derivative begins and ends at the zero point, passing through it at the absorbance band's maximum. Across the same wavelength, the upper side of this point exhibits a positive band, while the lower exhibits a negative band including both maxima as well as minima values; hence, this location is known as the inflection point.
The absorbance of a sample is discriminated against concerning wavelength to create the first, second, or higherorder derivatives (Figure 3).
A= f (λ): Zero order 
(13) 
dAdλ = f (λ): First order 
(14) 
d2Adλ2 = f (λ): Second order 
(15) 

a) 

b) 

c) 
Figure 3. Zeroth (a), first (b), and second (c) derivate spectra 
An absorption band's first derivative spectrum has a maximum, and a minimum, as well as, a crossover point at its λ_{max}. Finding the zero crossover point or wavelengths for each component is easily achieved with the use of the derived spectra. Absorbances of varying concentrations derived from stock solutions of separate components are measured at their corresponding zero crossover values acquired from their derivative spectra^{ }[7]. Regression analysis is carried out in conjunction with the plotting of calibration curves. The components are estimated by solving regression equations.
The derivative technique's key characteristics comprise increased information richness, differentiation against background noise, and more specificity in quantitative analysis^{ }[6].
Difference spectrophotometry
This method is based on the concept that between any two wavelengths, The concentration of the interest component on a mixed spectrum determines the absorbance difference (∆A), which is independent of the concentration of an interfering component given that the absorbance difference at the preferred wavelengths is zero^{ }[5]. Two wavelengths (λ_{1} & λ_{2}) are chosen for component X in a manner to ensure that the absorbance is the same at both wavelengths of interfering component Y. The calibration curves are obtained by plotting the absorbance difference (∆A) of each standard and sample mixture at λ1 and λ2 against the corresponding concentration. In the case of binary mixtures, the wavelength is chosen to ensure that the value of each component stands zero at the wavelength where the other components display maximum absorbance (Figure 4).

Figure 4. Individual absorption spectra of substances A and B; Difference absorption spectra C 
(Table 1) outlines several instances of different UV spectroscopic analytical methods in pharmaceutical applications.
Table 1. Applications of different UV analytical methods
Applications 
Method Used 
Ref. 
Acetaminophen and Chlorzoxazone 
Difference Spectrophotometry, QAbsorbance Method 
[8] 
Allopurinol and Lesinurad 
Simultaneous Equation Method 
[9] 
Ambroxol, Salbutamol, and Theophylline 
Simultaneous Equation Method 
[10] 
Bromfenac and Ofloxacin 
Derivative Spectrophotometry 
[11] 
Esomeprazole and Naproxen 
Derivative Spectrophotometry 
[12] 
Fluorescein and Benoxinate 
Simultaneous Equation Method 
[13] 
Fluticasone and Formoterol 
Simultaneous Equation Method, QAbsorbance Method 
[14] 
Furazolidone and Metronidazole 
QAbsorbance Method 
[15] 
Hydrochlorothiazide and Carvedilol 
QAbsorbance Method 
[16] 
Ledipasvir and Sofosbuvir 
Derivative Spectrophotometry 
[17] 
Levosulpiride and Rabeprazole sodium 
Derivative Spectrophotometry 
[18] 
Metformin HCl and Anagliptin 
QAbsorbance Method 
[19] 
Nalidixic acid and Metronidazole 
Difference Spectrophotometry 
[20] 
Pamabrom, Mefenamic Acid, and Dicyclomine Hydrochloride 
Simultaneous Equation Method 
[21] 
Quinfamide and Mebendazole 
QAbsorbance Method 
[22] 
Sofosbuvir and Velpatasvir 
Simultaneous Equation Method 
[23] 
Sumatriptan and Naproxen 
Simultaneous Equation Method 
[24] 
Telmisartan and Hydrochlorothiazide 
QAbsorbance Method 
[25] 
Tinidazole and Norfloxacin 
Difference Spectrophotometry 
[26] 
ΒCarotene and Lycopene 
Simultaneous Equation Method 
[27] 
Paracetamol
Paracetamol (PCM), widely known as Acetaminophen is an OTC medicine having analgesic and antipyretic properties used in mild to moderate pain and fever. It is chemically N(4hydroxyphenyl) acetamide (Figure 5). PCM comes under the category of nonsteroidal antiinflammatory drugs (NSAIDs).
It is considered to be a weak inhibitor of Prostaglandins (PGs). It works primarily by specifically inhibiting COX1 and COX2 through peroxidase's metabolizing activity (invivo). This results in inhibition in the formation of phenoxyl radical which is critical for prostaglandin production and cyclooxygenase activity of COX1, COX2.
The world's most commonly used pain reliever, recommended by the World Health Organization (WHO) as a firstline treatment drug in antiinflammatory therapy is Acetaminophen (paracetamol), commonly known as Tylenol. It is also used for its antipyretic properties, which help bring down a fever. Paracetamol is often found in combination with other medications in cold medicines, more than 600 overthecounter (OTC) allergy medicines, pain relievers, sleep aids, and other products.

Figure 5. Structures of drugs used with PCM in combination 
Estimation methods of paracetamol combinations
Paracetamol + Etodolac
Etodolac (ETO) is an NSAID with antipyretic and analgesic activity being used for chronic arthritis and acute pain. Its chemical name is 1,8Diethyl1,3,4,9tetrahydropyran (3,4b) indole1acetic acid. Similar to other NSAIDs, etodolac provides its antiinflammatory effect by inhibition of the enzyme cyclooxygenase (COX) preferably COX2 (about 550 times more selective than COX1). This results in the decrease of peripheral prostaglandins involved in mediating inflammation. Etodolac binds to the active site of the COX enzyme and prevents arachidonic acid from entering the active site.
A combination of 400mg Etodolac and 500mg Paracetamol is available in the tablet dosage form commercially. It has been found, from an extensive literature survey, that only a few UV spectroscopic and some RPHPLC methods are available for simultaneous estimation of this combination.
By taking Triethylammonium phosphate buffer as a solvent with the pH adjusted to 10 using 30% v/v orthophosphoric acid, Ashok Kumar, et al. (2015) utilized the simultaneous equation method of estimation [28]. The wavelength selected for ETO and PCM were 227nm and 252nm respectively. The developed method was validated for linearity which lay in the range of 515μg/ml for Etodolac and 6.2518.75μg/ml for Paracetamol.
In the ratio of 60:40 v/v as the common solvent for both drugs in the formulations, Alpa et al. (2013) and Shaikh et al. (2017) used methanol and water [29, 30]. The λ_{max} observed for the drugs were 247nm and 280nm for PCM and ETO respectively by Alpa et al. (2013) and 256nm and 286nm by Shaikh et al. (2017). The derivative spectroscopic method was used by both researchers with achieving zero cross points at 224.28nm and 219.27nm for Etodolac and Paracetamol respectively at Firstorder spectra out of the four derivatized. The method was validated for linearity, precision, and accuracy with concentration ranges of 525μg/ml (PCM) and 218μg/ml (ETO).
Balan et al. (2011) also used the simultaneous equation method for the estimation of the combination [31]. Phosphate buffer with pH 7.4 was used as the solvent instead of methanol. The maximum absorptive wavelength for PCM and ETO was found to be 242.5nm and 223.5nm respectively. The method was validated for linearity in the range of 210μg/ml for ETO and 214μg/ml for PCM.
Paracetamol + Diclofenac Sodium
Diclofenac Sodium (DIC) is an NSAID used in the condition of inflammation and acute and chronic pain with cases including osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. Diclofenac belongs to the family of phenylacetic acids having an analgesic, antipyretic and antiinflammatory activity^{ }[32]. DIC is a competitive, reversible, and nonselective inhibitor of cyclooxygenase (COX1 and COX2), which subsequently blocks the conversion of arachidonic acid to prostaglandin precursors. This inhibits the formation of prostanoids such as (PGE2) prostacyclin, and thromboxane, which are essential for response involved in pain, inflammation, and fever.
Paracetamol is a poorly watersoluble drug. From the literature study, it has been found that in the past years, a few Hydrotropic solubilization methods are used for simultaneous estimation with Diclofenac sodium.
Sharma et al. (2010) used 1.0 M Urea solution as a hydrotropic solubilizing agent to solubilize PCM for its spectrophotometric analysis [33]. Six methods in total in different studies were used. For the simultaneous equation method, the λ_{max} values of PCM and DIC were found to be 247nm and 276nm respectively. For Qanalysis, the isosbestic point was found to be at 268nm and λ_{max} of Diclofenac (276nm) was used as the second wavelength. Another method used was the Dual wavelength (Difference Spectroscopy) method. In this method, the Zerodifference wavelengths of PCM (245 and 249nm) and DIC (257 and 294nm) were selected for their estimation. The linearity range was within the range of 240μg/ml for both drugs.
In another study by Sharma et al, the Derivative spectroscopic method was used and calibration curves were plotted for PCM (240μg/ml) at 247nm and Diclofenac (240μg/ml) at 276nm [34]. For Area Under Curve Method (AUC), the regions selected (245249nm) for PCM and (276280nm) for DIC were used for the calculation of their concentrations. The aliquots were scanned at 247nm and 276nm and overlain spectra of mixed standards were obtained. The methods were validated for accuracy, precision, repeatability, and recovery study with standard deviation being <1.0% and RSD values being <2.0%. The linearity was within the concentrations selected.
Sharma et al. (2011) and Vandana Gupta et al. (2019) also used Urea as the Hydrotropic solubilizing agent in the concentrations 5M and 8M respectively [35, 36]. Sharma et al. (2011) used the simultaneous equation method with λ_{max }values being 247.8nm and 261.1nm for PCM and DIC respectively. The method was validated for accuracy, precision, repeatability, and recovery. The Beer’s law limit was found to be in the concentration range of 535μg/ml for both PCM and DIC.
Gupta et al. (2019) used the simultaneous equation method with λ_{max} at 243 and 276nm. In the Qanalysis method, the wavelengths selected were 264.4nm (λ_{1}isosbestic point) and 276nm. Which was further estimated by the Derivative spectrophotometric method in the First order derivative. The zero crossing points for PCM and DIC were 319.4nm and 276.8nm respectively. The methods were validated with %RSD value <1.0% in all three methods and a linearity limit between 525μg/ml.
Phaneemdra and Nagamalleswari (2012) used the firstorder derivative method with zero crossing points at 275.6nm (Diclofenac) used for Paracetamol and 242.69nm (Paracetamol) for Diclofenac [37]. Phosphate Buffer pH 6.8 was used as a common solvent. For the simultaneous equation method, the λ_{max} of observed at 243nm and 281nm. The linearity range was 210μg/ml and 525μg/ml for PCM and DIC respectively.
Ganesh et al. (2015) and Patel et al. (2020) used Distilled Water as a common solvent in determining the drug concentrations by the simultaneous equation method [38, 39]. The wavelengths selected were 247nm (PCM) and 276nm (DIC). Ganesh also used the QAbsorbance ratio method using the same solvent with selected wavelengths of 247nm and 265nm (isosbestic point). The proposed methods were validated for accuracy, linearity (630μg/ml), and precision with %RSD <2.0%.
Sebaiy et al. (2020) used the absorption subtraction method, ratio difference method, and derivative method. The solvent used is 90% Methanol [40]. For the advanced absorption subtraction method, the wavelengths were selected at 225nm (Isosbestic point) and 267nm (zero difference in absorbance of PCM). In the ratio difference method, selected wavelengths were 283nm and 270nm for Diclofenac and 251nm and 240nm for PCM. The firstorder derivative of the ratio difference curve was calculated and resulting spectra were measured at 273nm for DIC and 254nm for PCM. The absorption difference method is also incorporated by Chakravarthy et al. (2004) using methanol as solvent and the selected wavelengths at 230 and 254nm with zero absorbance difference for PCM and 260 and 292nm having zero difference for DIC [41].
In another study by Sebaiy et al., the HPoint assay method is used [42]. The wavelengths 225nm and 265nm were selected as zero difference points for PCM and shows a significant difference in absorption for DIC. The linearity was within the range of 7.54.5μg/ml for DIC and 422μg/ml for PCM in both studies. The correlation coefficient was found to be >0.9990 for both drugs and specificity values were 100.32% ± 0.51 for PCM and 100.25% ± 1.29 for Diclofenac.
Ibuprofen (IBU) is a commonly used NSAID that is considered to be one of the safest in the category. At low doses (8001,200 mg/day) it is approved for overthecounter sales and is generally safer to use. Ibuprofen is a derivative of propionic acid that has antiinflammatory, analgesic, and antipyretic properties because it inhibits cyclooxygenase I and II nonselectively, which reduces prostaglandin production, by prostaglandin synthase, the main physiologic effect of ibuprofen. Ibuprofen can also inhibit platelet aggregation by decreasing the formation of thromboxane A2.
From an extensive literature survey, it has been found that various methods and approaches have been used for the simultaneous determination of PCM and IBU in the combined dosage form. The simultaneous equation method is used by Gondalia et al. (2010) for combination drugs present in soft gelatine capsule dosage form [43]. Methanol was used as a common solvent and the wavelengths selected were 224nm and 248nm. The method was validated for linearity which was found to be in the range of 414μg/ml (IBU) and 212μg/ml (PCM), and accuracy with a %recovery of 99.70 ± 1.08 and 100.16 ± 1.02 for IBU and PCM, respectively. %RSD values were 1.44 and 0.95 for the same.
Harshini et al. (2014) and Gaikwad et al. (2017) also used the simultaneous equation method with different solvents i.e., Ethanol and 0.1N NaOH respectively [44, 45]. In both studies, the λ_{max} of PCM and IBU were found to be at 240nm and 220nm. The developed methods were validated with linearity in the range of 220μg/ml for IBU and 115μg/ml for PCM.
Tejashree et al. (2020) used Methanol as a common solvent for both drugs [46]. For the simultaneous equation method, the wavelengths selected were 256nm and 222.4nm as λ_{max} of PCM and IBU respectively. 226.4nm was observed as the isoabsorptive point for the Qanalysis method. 530μg/ml was the linearity concentration range for both drugs. The recovery study resulted in the values 102.65% for PCM and 100.83% for IBU. %RSD values were 0.58 and 0.47.
Ostwal et al. (2012) and T. Mamatha et, al. (2013) used the dissolution method using Phosphate buffer (pH 5.8 and 7.2 respectively) as the dissolution medium [47, 48]. The wavelengths selected were 222.4nm (λ_{max} IBU) and 226.4nm (Isoabsorptive point) by Ostwal and 221.8nm and 213.8nm by Mamatha for estimation by absorbance ratio method using the concentration range within the linearity limit of 221μg/ml for IBU and 214μg/ml for PCM.
Hassan, (2008) used chemometric methods including ratio derivative and multivariate methods (Classical Least Square and Principal components regression analysis) for simultaneous determination of the drug combination [49]. Methanol was used to prepare the aliquots, 290nm and 230nm were observed as zerocrossing points for IBU and PCM, respectively. For the first derivative, the amplitudes measured at 280nm and 270nm were found linear to the concentrations of IBU and PCM, respectively. For multivariate analysis, ten solutions were prepared with a linearity concentration range of 560 and 10100μg/ml for ibuprofen and paracetamol, respectively. The Calibration K matrix was obtained from the absorption data in the range of 10040nm. The methods were validated for accuracy, precision, and repeatability with %RSD values being within the range.
The ratio spectra method is also used in another development by Zayed et al. (2011) with getting Mean recovery % of 96.83(IBU) and 97.59(PCM) in the first derivative; and 97.16(IBU) and 96.62(PCM) in the second derivative spectra [50]. The linearity was found between the range of 232 (IBU) and 224μg/ml (PCM). The same solvent was used as in the previous study.
Another study by Hoang et al. (2014) also used derivative spectroscopy along with wavelet transforms [51]. Phosphate buffer pH 7.2 was used as the solvent. 249.3 and 242.0 nm were observed as zerocrossing points for IBU and PCM, respectively. The beer’s law limit was within the concentration range of 1232μg/ml (IBU) and 2040μg/ml (PCM). The spectrophotometric results were found to be 95% accurate when statistically compared with the HPLC method taken as standard.
Omray et al. (2007) used the absorbance difference method for the simultaneous determination of the combination [52]. Ethanol was used as a common solvent. Absorbance was scanned over a range of 200 – 600 nm. Two wavelengths 220 and 231nm were selected with absorbance difference for IBU being zero. Similarly, 241 and 255nm were selected for having zero absorbance difference for PCM. The method was validated in terms of linearity (612μg/ml), accuracy, precision, specificity, and reproducibility of the sample applications.
ElMaraghy and Lamie (2019) also used the ratio difference method for the resolution of overlapped zeroorder spectra [53]. Methanol was used as a common solvent to achieve a concentration of 220μg/mL for PCM and 250μg/mL for IBU which was proven for linearity. The zeroorder spectra were measured over the range of 200400nm. Two wavelengths each with a maximum difference in peak amplitudes for PCM (236 and 248 nm) and IBU (210.6 and 216.4 nm) were selected and a calibration curve was plotted. %RSD was found to be 0.650 and 0.778; and the Mean recovery% values were 99.91 and 100.18 for PCM and IBU, respectively.
Paracetamol + Domperidone
Domperidone (DOM) is a dopamine antagonist with antiemetic, gastrokinetic, and galactagogue activities. It binds to the D2 receptor in the chemoreceptor trigger zones which inhibits dopamine binding and D2Rmediated signaling affecting the motor functions of the GIT and relieving various gastrointestinal (GI) symptoms, such as nausea and vomiting.
A literature survey has revealed that only a few validated methods have been developed for simultaneous estimation of Paracetamol and Domperidone as a combination drug therapy from the year 2009 till 2016 and no recent development has taken place since.
Kapil et al. (2009) used the simultaneous equation method for the determination [54]. Methanol is used as a common solvent and the λ_{max} was measured at 250nm and 285nm for PCM and DOM, respectively. The method was validated for accuracy, precision, specificity, and ruggedness with recovery study values found to be 99.45±0.47% for PCM and 100.67±0.18 for DOM. The linearity range was observed to be 530μg/ml (PCM) and 0.85μg/ml (DOM).
Babar et al. (2012) used two simple methods, the simultaneous equation method with wavelengths selected were 243.4 nm and 284.12 nm as the corresponding λ_{max} of PCM and DOM [55]. For the absorption ratio method, 270nm was recorded as the absorptive point of the two drugs. The method was validated and recovery studies were performed with linearity concentrations to be found in the range between 1530μg/ml (DOM) and 1116μg/ml (PCM).
Appasaheb et al. (2013) also used the simultaneous equation method taking 258nm and 292nm as maximum absorption wavelengths for PCM and DOM, respectively. 0.1N NaOH was taken as a common solvent [56]. The dual wavelengths with zero absorbance difference for DOM (247nm and 269nm) and PCM (288nm and 296nm) were selected for the absorbance difference method. Another method developed was Area under curve method with sampling wavelength ranges selected 242nm275nm for PCM and 284nm302nm for DOM from the calibration curve. The methods were validated for accuracy and precision with obtaining linearity concentration between the range 530μg/ml for both PCM and DOM.
Mali et al. (2016) also used the AUC method for the simultaneous determination of the combination drugs with a wavelength range of 220274nm for Paracetamol and 262304nm for Domperidone from the calibration curve [57]. The maximum wavelengths 248nm (PCM) and 286nm (DOM) were used to plot the calibration curve by simultaneous equation method. In a separate study [58], Mali A. used the First order derivative overlain spectra for further resolution of the zeroorder spectrum overlapping. The zero crossing points 262nm (PCM) and 297nm (DOM) were used to measure the firstorder derivative values of paracetamol and domperidone, respectively. Both studies revealed that the linearity for both drugs was observed in the range of 525μg/ml by all three methods.
Paracetamol + Aceclofenac
Aceclofenac (ACF) is a Phenylacetic acid derivative that is the carboxymethyl ester of Diclofenac. It is NSAID with antiinflammatory and analgesic properties and is used in the management of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, low back pain (LBP), scapulohumeral periarthritis, extraarticular rheumatism, odontalgia. It is reported to have higher Antiinflammatory action and is well tolerated with a more favorable GI profile than other NSAIDs.
From a comprehensive literature survey, it has been found that several methods have been developed for the estimation of the combination of Aceclofenac and Paracetamol in the last two decades including Viedort's method, Qanalysis, and Ratio derivative method.
Mishra and Garg (2006) used the simultaneous equation method and Qanalysis method by taking Ethanol as a common solvent [59]. The absorption maxima of PCM and ACF were observed at wavelengths 256nm and 275nm, respectively. 230nm was observed as the isosbestic point for the two drugs. The method showed linearity within the concentration range of 110μg/ml. The recovery study was well within the range of 99100%.
Pawar et al. (2010) also used the simultaneous equation method utilizing 274nm and 248nm as the estimation wavelengths for PCM and ACF respectively [60]. Methanol: Glass distilled water was used as the common solvent. The linearity concentrations were within the range of 15μg/ml (ACF) and 525 (PCM).
Jain et al. (2007) and Gharge et al. (2010) also used the same abovementioned vierodt’s and Qanalysis methods but with Methanol (pure and 80%, respectively) as a common solvent [61, 62]. The wavelengths selected were 249nm, 276nm, and 270nm [61]; 245nm, 276nm, and 267.5nm [62]. The Linearity concentrations observed for PCM and ACF were 225μg/ml and 130μg/ml respectively by Jain A.; and 220μg/ml and 540μg/ml by Gharge D.
Mahapare et al. (2007) used the Difference spectroscopy method and AUC method for determination using ARgrade Methanol as the solvent [63]. 274.5nm and 244nm were the selected wavelengths (λ_{max} of ACF and PCM). For AUC overlain spectrum was obtained and the concentrations were measured using the selected wavelength ranges, 224 to 260 nm (ACF) and 254 to 294 nm (PCM). For the absorption difference method, the wavelengths selected were 221.5nm and 257nm for ACF and, 261nm and 278nm for PCM. The methods were validated in terms of linearity of absorbance in the concentration range of 220 μg/ml (ACF) and 540 μg/ml (PCM) at their respective maxima.
The absorbance difference method was also implied by Pradhan et, al. (2019) using the same solvent as above. 245nm and 214nm were observed as the absorbance maxima of PCM and ACF respectively [64]. The wavelengths selected from the spectrum were 245 and 270nm for the estimation of PCM and for the estimation of ACF wavelengths 214 and 242nm were chosen as λ_{1} and λ_{2}. The range for linearity was found to be 340μg/ml for PCM and 310μg/ml for ACF.
Gandhi et al. (2008) used the Ratio Derivative method with selected wavelengths, 256nm (PCM) and 268nm (ACF) from the firstorder derivative spectra [65]. Linearity was found in the range of 1050μg/ml with high correlation coefficients for both the drugs and %RSD <1.5.
A similar method, The Firstorder derivative method was used by Nimbekar et al. (2014) with zerocrossing points observed at 276nm for ACF and 248nm for PCM [66]. vierodt’s method was also implied using the respected absorbance maxima. The linearity was found to be in the range, of 330μg/ml (PCM) and 220μg/ml (ACF).
Kumar et al. (2011) and Mishra et al. (2014) used the Qanalysis absorbance ratio method using wavelengths 275.4nm (λ_{max} ACF) and 266.1nm (isosbestic point); and 268nm (isosbestic) and 238nm (λ_{max} PCM), respectively [67, 68]. In the former study, the linearity range was achieved between 135µg/ml for ACF and 115µg/ml for PCM. Ganesh Mishra also used the derivative method with zerocrossing points at 238nm (ACF) and 268nm (PCM) in the firstorder derivative spectra. The linearity found between the concentration range is 550mg/ml. Both studies showed good recovery within the range of 99102% and %RSD <2%.
Caffeine (CAF) is a CNS stimulant methylxanthine alkaloid, structurally related to adenosine, and primarily acts as an adenosine receptor antagonist. It has psychotropic and antiinflammatory activities with increased energy metabolism throughout the brain but induced brain hypoperfusion. It reduces myocardial blood flow and limits adenosinemediated vasodilation by inhibiting A1, A2A, and A2B adenosine receptors in blood vessels. The antiinflammatory effect is caused due to competitive inhibition of PDE (Phosphodiesterase) which leads to an increase in the amount of cAMP, protein kinase activation, and inhibited leukotrienes synthesis which ultimately assists in reducing inflammation.
Paracetamol and caffeine as a combination act as a good analgesic and antipyretic drug therapy. During the last two decades, several methods have been developed for the estimation of the combination simultaneously by UV spectrophotometer. Due to huge variables and a large number of absorbance values, chemometricassisted methods have been preferred for rapid and precise estimation.
Multivariate methods like principal component regression (PCR), partial leastsquares regression (PLS), and artificial neural networks (ANN) were used by Dinç & Baleanu (2002); and Aktaş and Kitiş, (2014)^{ }[69, 70]. Dinc and Baleanu measured the absorbances at an interval of 15λ in the region of 215 – 285 nm. 0.1 M HCl was used as the common solvent. ‘MAPLE V’ software was used for solving complex regression equations. Aktaş and Kitiş used ‘Minitab 16’ software using the same 0.1 N HCl as a common solvent. The absorption spectra were measured in the spectral region 205305nm with a much smaller ∆λ value, set to 0.1nm.
In a more recent study by Karim et al, (2019) Partial least square regression and artificial neural network methods are used for the simultaneous essay of PCM and CAF [71]. The spectra region 205300nm was used for recording the absorbance with an interval of 1nm and preferred common solvent Methanol. The software ‘MATLAB 2014’ and ‘Unscrambler® X’ has been used for ANN and PLS respectively. Both drugs showed an R^{2} value of 99.28% for prediction and 99.13% for the validation set.
Tavallali and Sheikhaei, (2009) used the Hpoint standard addition method for the simultaneous estimation of the drug combination [72]. The wavelength used is of the visible region; 453nm. Acetic acid buffer pH 5.0 is used as the reagent for the essay. The linearity was within the range of 0.13μg/ml for CAF and 1.57μg/ml for PCM.
Vichare et al., (2010) used the simpler simultaneous equation method and absorption ratio method for the estimation of the combination [73]. 243nm and 273nm were observed as λ_{max} of PCM and CAF, respectively and wavelength 259.5nm was the isosbestic point. The stocks were prepared by dissolving the drugs in distilled water. 232 and 216μg/ml were the linearity range for CAF and PCM respectively.
Sharma et al. (2015) used the Dual wavelength method with selected wavelengths 260nm and 281nm for PCM and 234nm and 249nm for CAF [74]. Methanol was taken as solvent. The linearity concentration ranges were 1060 and 318 μg/mL for paracetamol and caffeine, respectively.
Aspirin (ASP) also known as acetylsalicylic acid is an orally administered NSAID most widely used in the condition of pain, fever, myocardial infarction, osteoarthritis, and ischemia [75].
It has antiinflammatory and antipyretic activity caused by nonselective inhibition of COX leading to lowered prostaglandin levels. Unlike other NSAIDs, it binds irreversibly to COX II and also blocks thromboxane A2 on platelets, preventing platelet aggregation [76].
From an exhaustive literature survey, it has been found that only a couple of studies have been performed for simultaneously estimating aspirin and paracetamol in the combined dosage form.
Samnani et al. (2007) used Vierordt’s method for the determination of aspirin and paracetamol in treated sewage water [77]. Double Distilled Water (DDW), Methanol, and 0.1N HCl were used to prepare separate stock solutions for both drugs. The wavelength used for recording the absorbance was 225nm for ASP and 244nm for PCM. The results were compared to that of HPLC. The method was validated for linearity, precision, and accuracy with %RSD less than 0.008 for both drugs and correlation coefficient being 0.9626 (ASP) and 0.9989 (PCM).
Murtaza et al. (2010) also used the simultaneous equation method with selected wavelengths 265nm and 257nm as λ_{max} of ASP and PCM respectively [78]. The solvent was prepared by mixing 0.1N HCl and Methanol in equal parts. The linearity was between the concentration range of 2 to 64µg/ml.
Piroxicam (PIR) is an NSAID of the oxicam class used for its antiinflammatory, antipyretic, and analgesic activity. Piroxicam nonselectively bind to cyclooxygenase enzymes inhibiting prostaglandin synthesis. It reversibly stops the conversion of arachidonic acid into prostaglandin precursors which leads to inflammation. It is used to treat chronic ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, softtissue disorders, acute gout, and also in postoperative pain^{ }[79].
Not a lot of methods have been developed for this combination of drugs. It’s been revealed that only two studies have been performed so far regarding the same.
Shirkhedkar et al. (2008) used the QAbsorbance method with selected wavelengths 257nm (λmax of PCM) and 320nm (the absorptive point) [80]. 0.01N NaOH was used as the common solvent for dissolving both drugs [81]. The linearity range was 412µg/ml and 440µg/ml.
In a more recent study, the chemometric Partial least square method has been implied by Pretty Falena Atmanda Kambira et al. (2020). 0.1N NaOH was used as a common solvent. A wavelength range of 200500nm (UVVisible combined) was used for recording the absorbance with an interval of 1nm [82]. Software ‘UV Probe v2.52’ was used for interpreting the data. The Root mean square of error crossvalidation (RMSEC) values are 0.125 and 0.087.
CONCLUSION
At present, various analytical methods are available for the simultaneous estimation of combination drugs, yet further studies regarding the same should be performed to develop newer, simpler, economic, and robust methods with good linearity and recovery. UVvisible spectroscopy offers a straightforward, less timeconsuming, accurate, and very sensitive approach for estimating various medication combinations for which no method of estimation has yet been published.
This compilation study will provide the researchers working in the field with extensive knowledge and data about the already developed UV spectroscopic methods and will assist them further in their research (Table 2).
Table 2. Estimation examples of different combinations of paracetamol
S. NO. 
STUDIES 
METHOD USED 
WAVELENGTH (nm) 
LINEARITY LIMIT (μg/ml) 
SOLVENT USED 

λ1 
λ2 
DRUG 
PCM 

PARACETAMOL + ETODOLAC 

1 
Shailaja et al. (2015) [28] 
Simultaneous Equation Method 
252 
227 
5.0015.00 
6.2518.75 
Triethylammonium phosphate buffer pH 10 
2 
Alpa et al. (2013) [29] 
Derivative Spectroscopic Method 
280 
247 
2.0018.00 
5.0025.00 
Methanol and water (60:40) 
3 
Saikh et al. (2017) 
Derivative Spectroscopic Method 
280 
247 
2.0018.00 
5.0025.00 
Methanol and water (60:40) 
4 
Balan et al. (2011) [31] 
Simultaneous Equation Method 
223.5 
242.5 
2.0010.00 
2.0014.00 
Phosphate buffer pH 7.4 
PARACETAMOL + DICLOFENAC SODIUM 

1 
Sharma et al. (2010) [33] 
Simultaneous Equation Method 
247 
276 
240 
240 
1.0 M Urea 
QAnalysis 
268 
276 

Difference Spectroscopy 
259, 294 
245, 249 

2 
Jain & Sharma, (2010) [34] 
Derivative Spectroscopy 
247 
276 
240 
240 
1.0 M Urea 
Area Under Curve 
245249 
276280 

Multicomponent Method 
247 
276 

3 
Sharma et al. (2011) [35] 
Simultaneous Equation Method 
247.8 
261.1 
535 
535 
5 M Urea 
4 
Gupta et al. (2019) [36] 
Simultaneous Equation Method 
243 
276 
525 
525 
8 M Urea 
QAnalysis 
264.4 
276 

Derivative Spectroscopy 
243 
276 

5 
Phaneemdra & Nagamalleswari (2012) [37] 
Derivative Spectroscopy 
275.6 
242.69 
210 
525 
Phosphate Buffer pH 6.8 
Simultaneous Equation Method 
243 
281 

6 
Ganesh et al. (2015) [38] 
Simultaneous Equation Method 
247 
276 
630 
630 
Distilled Water 
QAnalysis 
247 
265 

7 
Patel et al. (2020)[39] 
Simultaneous Equation Method 
247 
276 
630 
630 
Distilled Water 
8 
Sebaiy et al. (2020) [40] 
Absorption Subtraction 
227 
267 
7.545 
422 
Methanol 90% 
Difference Spectroscopy 
283, 270 
251, 240 

Derivative Spectroscopy 
273 
254 

9 
Sebaiy et al. (2020) [42] 
HPoint Essay 
225 
265 
7.545 
422 
Methanol 90% 
10 
Saheb et al. (2004) [41] 
Difference Spectroscopy 
230, 254 
260, 292 
Methanol 

PARACETAMOL + IBUPROFEN 

1 
Gondalia et al. (2010) [43] 
Simultaneous Equation Method 
224 
248 
414 
212 
Methanol 
2 
Harshini et al. (2014) [44] 
Simultaneous Equation Method 
240 
220 
220 
115 
Ethanol 
3 
Gaikwad et al. (2017) [45] 
Simultaneous Equation Method 
240 
220 
250 
280 
0.1 N NaOH 
4 
Tejashree et al. (2020) [46] 
Simultaneous Equation Method 
256 
222.4 
530 
530 
Methanol 
QAnalysis 
256 
226.4 

5 
Ostwal et al. (2012) [47] 
QAnalysis 
222.4 
226.4 
Phosphate Buffer pH 5.8 

6 
Tirunagari et, al. (2013) [48] 
QAnalysis 
221.8 
213.8 
221 
214 
Phosphate Buffer pH 7.2 
7 
Hassan (2008) [49] 
Derivative Spectroscopy 
230 
290 
5100 
10100 
Methanol 
8 
Hoang et al. (2014) [50] 
Derivative Spectroscopy 
249.3 
242 
1232 
2040 
Phosphate Buffer pH 7.2 
9 
Omray et al. (2007) [52] 
Difference Spectroscopy 
220, 231 
241, 255 
Ethanol 

10 
ElMaraghy & Lamie (2019) [53] 
Difference Spectroscopy 
210.6, 216.4 
236, 248 
250 
220 
Methanol 
PARACETAMOL + DOMPERIDONE 

1 
Kapil et al. (2009) [54] 
Simultaneous Equation Method 
250 
285 
0.85 
530 
Methanol 
2 
Babar et al. (2012) [55] 
Simultaneous Equation Method 
243.4 
284.12 
1530 
1116 
Methanol 
QAnalysis 
243.4 
270 

3 
Appasaheb et al. (2013) [56] 
Simultaneous Equation Method 
258 
292 
530 
530 
0..1 N NaOH 
Difference Spectroscopy 
247, 269 
288, 296 

Area Under Curve 
284302 
242275 

4 
Mali et al. (2016) [57] 
Area Under Curve 
262304 
220274 
525 
525 
Methanol 
Simultaneous Equation Method 
286 
248 

Derivative Spectroscopy 
262 
297 

PARACETAMOL + ACECLOFENAC 

1 
Mishra & Garg (2006) [59] 
Simultaneous Equation Method 
275 
256 
110 
110 
Ethanol 
QAnalysis 
275 
230 

2 
Pawar et al. (2010) [60] 
Simultaneous Equation Method 
274 
248 
15 
525 
Methanol 
3 
Jain et al. (2007) [61] 
Simultaneous Equation Method 
276 
249 
130 
225 
Methanol 
QAnalysis 
276 
270 

4 
Gharge et al. (2010) [62] 
Simultaneous Equation Method 
276 
245 
540 
220 
Methanol 80% 
QAnalysis 
276 
267.5 

5 
Mahaparale et al. (2007) [63] 
Difference Spectroscopy 
221.5, 257 
261, 278 
220 
540 
Methanol 
Area Under Curve 
224260 
254294 

6 
Basnett et al. (2019) [64] 
Difference Spectroscopy 
214, 242 
245, 270 
310 
340 
Methanol 
7 
Nikam et al. (2008) [65] 
Derivative Spectroscopy 
268 
256 
1050 
1050 
Methanol 
8 
Chaudhari et al. (2014) [66] 
Simultaneous Equation Method 
276 
248 
220 
330 
Methanol: Distilled Water 
Derivative Spectroscopy 
276 
248 

9 
Kumar et al. (2011) [67] 
QAnalysis 
275.4 
266.1 
135 
115 
Methanol 
10 
Mishra et al. (2014) [68] 
QAnalysis 
268 
238 
550 
550 
2 M Urea & 5 M Sodium Acetate (20:30) 
Derivative Spectroscopy 
238 
268 

PARACETAMOL + CAFFEINE 

λ Range 
∆λ 
DRUG 
PCM 

1 
Aktaş and Kitiş, (2014) [70] 
Principal component regression (PCR), 
205305 
0.1nm 
 
 
0.1 N HCl 
Partial leastsquares regression (PLS), 

Artificial neural networks (ANN) 

2 
Dinç & Baleanu (2002) [69] 
PCR, 
215285 
15nm 
 
 
0.1 M HCl 
PLS 

3 
Uddin et al, (2019) [71] 
PLS, 
205300 
1nm 
 
 
Methanol 
ANN 

λ1 
λ2 
DRUG 
PCM 

4 
Tavallali & Sheikhaei, (2009) [72] 
HPoint Essay 
453 
453 
0.13.0 
1.57.0 
Acetic Acid Buffer pH 5 
5 
Vichare et al., (2010) [73] 
Simultaneous Equation Method 
243 
273 
232 
216 
Distilled Water 
QAnalysis 
243 
259.5 

6 
Sharma et al. (2015) [74] 
Difference Spectroscopy 
234, 249 
26, 281 
318 
1060 
Methanol 
PARACETAMOL + ASPIRIN 

1 
Samnani et al. (2007) [77] 
Simultaneous Equation Method 
225 
244 
Double Distilled Water (DDW) 

Methanol 

0.1N HCl 

2 
Ghulam et al. (2010) [78] 
Simultaneous Equation Method 
257 
265 
264 
264 
0.1N HCl + Methanol (1:1) 
PARACETAMOL + PIROXICAM 

1 
Shirkhedkar et al. (2008) [80] 
QAnalysis 
320 
257 
440 
412 
0.01N NaOH 
2 
Kambira et al. (2020) [82] 
PLS 
200 to 
500 
 
 
0.1N NaOH 
ACKNOWLEDGMENTS : The authors thank GLA University, Mathura, UP, for the providing necessary facilities.
CONFLICT OF INTEREST : None
FINANCIAL SUPPORT : None
ETHICS STATEMENT : None
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