Comparative in vitro dissolution studies of fixed-dose combination formulations: analgesic tablets of acetaminophen/caffeine

A simple and rapid UV derivative method with zero-crossing determinations was developed for estimation of acetaminophen (ACE) and caffeine (CAF) in fixed-dose combination formulations. The first-derivative of standard solutions of both drugs were used and ACE and CAF were quantified at 273.0 and 216.5 nm, respectively. The method was validated, and it was applied to dissolution studies with the USP Apparatus 2 and flow-through cell (USP Apparatus 4). Dissolution profiles comparisons (generic vs reference) were carried out with model-independent and model-dependent approaches. Mean dissolution time and dissolution efficiency were calculated and significant differences, in almost all calculated parameters, were found (p<0.05). Weibull, logistic, Gompertz, and Probit models were used to fit dissolution data and Probit was the best-fit model that describes the in vitro dissolution performance of ACE and CAF. Using t50% data, derived from this fit, dissolution profiles of ACE in USP Apparatus 2 were significant different (p<0.05). The proposed UV derivative method generates reliable information that can be compared with published results. Dissolution studies of fixed-dose combination formulations are important because quality of generic drug products depends on quality of references. It is essential to maintain a post-marketing evaluation of formulations with analgesic drugs mixed with CAF to offer the population high quality medicines.

A simple and rapid UV derivative method with zero-crossing determinations was developed for estimation of acetaminophen (ACE) and caffeine (CAF) in ixed-dose combination formulations. The irst-derivative of standard solutions of both drugs were used and ACE and CAF were quanti ied at 273.0 and 216.5 nm, respectively. The method was validated, and it was applied to dissolution studies with the USP Apparatus 2 and low-through cell (USP Apparatus 4). Dissolution pro iles comparisons (generic vs reference) were carried out with model-independent and model-dependent approaches. Mean dissolution time and dissolution ef iciency were calculated and signi icant differences, in almost all calculated parameters, were found (p<0.05). Weibull, logistic, Gompertz, and Probit models were used to it dissolution data and Probit was the best-it model that describes the in vitro dissolution performance of ACE and CAF. Using t 50% data, derived from this it, dissolution proiles of ACE in USP Apparatus 2 were signi icant different (p<0.05). The proposed UV derivative method generates reliable information that can be compared with published results. Dissolution studies of ixed-dose combination formulations are important because quality of generic drug products depends on quality of references. It is essential to maintain a post-marketing evaluation of formulations with analgesic drugs mixed with CAF to offer the population high quality medicines.

INTRODUCTION
Acetaminophen (ACE) has antipyretic, analgesic, and weak anti-in lammatory actions, it is sparingly soluble in water and prone to dissolution and bioavailability problems (Hamed et al., 2017). Due to its high solubility and low permeability ACE is considered as a class III drug (Kalantzi et al., 2006). Caffeine (CAF) is a stimulant of the central nervous system (Sawynok and Yaksh, 1993) and it has been added to non-opioid drugs to improve the analgesic effects (López et al., 2006). Chemical structures of ACE and CAF are shown in Figure 1.
Dissolution studies are usually carried out with USP basket and paddle apparatuses (USP Apparatus 1 and 2, respectively). Dissolution test of ixed-dose combination formulations of ACE/CAF is described in the USP (United States Pharmacopeial Convention, 2019). The USP Apparatus 2 at 100 rpm with 900 ml of water and chromatographic (HPLC) determination should be used. Several authors have pointed out that slow agitation rates are preferable to develop a discriminative dissolution method (Shah et al., 1992). By the previously reported information there is a monograph that suggests the waiver of bioequivalence studies of ACE tablets by in vitro dissolution studies (Kalantzi et al., 2006) however, for ixed drug combination products where class I or III drugs are combined with any other class of drug a biowaiver approach is not applicable (FDA, 2017).

Figure 1: Chemical structure of acetaminophen (left) and caffeine (right)
The low-through cell method (USP Apparatus 4) has many advantages to study the in vitro dissolution performance of active pharmaceutical ingredients (API) under a particular hydrodynamic environment (Looney, 1997;Brown, 2005). Several authors have reported in vitro/in vivo correlation with dissolution methods where the USP Apparatus 4 is used (ichi Jinno et al., 2008;Jantratid et al., 2009;Fang et al., 2010). Despite the advantage of low-through cell to study the dissolution performance of drugs, only information about in vitro release of ACE/CAF from nano ibers and a modi ied dissolution procedure (Illangakoon et al., 2014) as well as with pharmaceutical formulations and the USP Apparatus 1 (Williams et al., 2010) has been reported.
Analytical methods to quantify drug mixtures in dissolution studies are mainly based on HPLC determinations however, HPLC methods are not friendly to the environment due to the high generation of toxic waste in addition to the use and maintenance of expensive equipment. UV derivative methods are an ef icient alternative to identify and quantify mixtures of two or more drugs in a similar way as HPLC methods. Some UV derivative methods have been developed to dissolution studies of ixed-dose combination formulations (Medina et al., 2013(Medina et al., , 2016Medina-López et al., 2020).
Generic drugs are off-patent formulations that contain the same API, and the same dose, as reference drug product (Ruiz et al., 2012). These formulations must demonstrate the same quality and ef icacy as the reference products. A variety of comparative in vitro dissolution studies with generic tablets of only one API have recently been published (Varillas et al., 2020;Usman et al., 2020) whereas that some authors have reported the comparison of generic drugs and reference with not entirely favorable results (Akdag et al., 2020;Alvarado et al., 2020). Dissolution studies with mixtures of non-steroidal anti-in lammatory drugs (NSAIDs) and CAF are scarce.
The aim of this study was to develop a UV derivative method with zero-crossing determinations to quantify ACE and CAF in ixed-dose combination formulations and to use the analytical procedure in a comparative in vitro dissolution study of analgesic tablets. The USP Apparatuses 2 and 4 with 0.1 M phosphate buffer pH 7.4, as dissolution medium, were used. Dissolution pro iles of reference and a generic formulation were compared by modelindependent and model-dependent approaches. By knowing the release performance of reference, it is possible to design better generic formulations.

Chemicals
ACE and CAF standards were purchased from Sigma-Aldrich Co. (St. Louis MO, USA). Sodium phosphate monobasic and dibasic crystals were purchased from J.T.Baker-Mexico (Xalostoc-Mexico). Two ACE/CAF ixed-dose combination formulations (500/50 mg tablets) were used in this study. One of them was the Saridon ® product (Bayer de México S.A. de C.V., Mexico). Mexican health regulatory agency (Cofepris) has established this commercial brand as the reference to be used in bioequivalence studies (Cofepris, 2020). The second drug product was a generic formulation. The R and G letters were assigned to them, respectively.

Spectrophotometric conditions
ACE and CAF were simultaneously determined by a proposed UV derivative method. A double beam UV/Vis spectrophotometer (Perkin Elmer Lambda 35, Waltham MA, USA) with 1 cm quartz cells was utilized. The operating conditions for UV analysis were irst-derivative mode ( 1 D) with scan speed 240 nm/min, slit width 2 nm and sampling interval 1 nm.

Standard calibration curves
Stock solutions of ACE and CAF in 0.1 M phosphate buffer pH 7.4 (1 mg/ml) were separately prepared. Five standard solutions of each drug in 0.1 M phosphate buffer pH 7.4 were prepared.
The concentration range of ACE was 10-30 µg/ml and for CAF was 0.5-5 µg/ml. Then, the zero order spectra taken from 200 to 350 nm, using 1 cm quartz cells, were recorded, and stored. With these spectra the 1 D of each solution was calculated.

Linearity, precision, and accuracy
Linearity was tested with the preparation of two standard calibration curves of each drug. All solutions were analyzed according to the procedures described above and mean data were used for mathematical treatments. Linear regression analysis was calculated and an analysis of variance (ANOVA) of each regression was carried out. The 95% con idence intervals (CI 95% ) for intercepts were calculated. Precision was veri ied with the calculation of coef icient of variation (CV) at each concentration level. Accuracy was con irmed with the analysis of three synthetic mixtures at following concentrations: 12, 18, and 27 µg/ml of ACE and 1.5, 3, and 4 µg/ml of CAF. Mixtures were analyzed according to the proposed UV derivative method. Found vs. added concentrations were plotted and linear regression analysis were calculated. Then, CI 95% for slopes and intercepts were determined.

Uniformity of dosage units and assay tests
Both tests were performed to R and G formulations according the USP procedures (United States Pharmacopeial Convention, 2019).

Dissolution pro iles USP Apparatus 2
Dissolution pro iles of ACE and CAF were obtained in a USP Apparatus 2 (Sotax AT-7 Smart, Switzerland) at 75 rpm using 900 ml of 0.1 M phosphate buffer pH 7.4 at 37.0±0.5 • C as dissolution medium. Prior to use, the dissolution medium was deareated by vaccum and transferred into the dissolution vessels. At 10, 20, 30, 45, and 60 min a volume of 3 ml was withdrawn, and it was iltered with 0.45 µm nitrocellulose ilters (Millipore ® ). The amounts of ACE and CAF dissolved were determined with standard calibration curves of each drug.

USP Apparatus 4
Dissolution pro iles of ACE and CAF were obtained in a USP Apparatus 4 (Sotax CE6, Sotax AG, Switzerland) with 22.6 mm cells (i.d.). Laminar low (generated with a bed of 6 g of glass beads) was used.
The degassed dissolution medium, 0.1 M phosphate buffer pH 7.4 at 37.0±0.5 • C was pumped at a low rate of 16 ml/min. An open system was used, without recycling the dissolution medium. At 10, 20, 30, 45, and 60 min a volume of 3 ml was withdrawn, and it was iltered with nitrocellulose ilters. For every trial, standard calibration curves were prepared.

Data analysis
Dissolution pro iles of ACE and CAF from R and G formulations were compared by modelindependent and model-dependent approaches.
For model-independent comparisons f 2 similarity factor, mean dissolution time (MDT), and dissolution ef iciency (DE) were calculated. Similarity factor was calculated according to equation previously published (Moore and Flanner, 1996). Similar dissolution pro iles were considered if f 2 =50 to 100. MDT and DE were compared by a Student's t-test and signi icant differences were considered if p<0.05. For model-dependent comparisons, dissolution data were itted to Hyperbola equation and with a and b parameters t 50% values were calculated. Additionally, dissolution data of ACE and CAF were itted to Weibull, logistic, Gompertz, and Probit models (Zhang et al., 2010). Mathematical equation of each model is shown in Table 1. The model with the highest adjusted determination coef icient (R 2 adjusted ) and lowest Akaike Information Criterion (AIC) was chosen as the best-it model (Yuksel et al., 2000). Data analysis was carried out using the Excel add-in DDSolver program (Zhang et al., 2010).

UV derivative method
The adequate identi ication and quanti ication of ACE and CAF with the proposed UV derivative method is shown in Figure 2.
As shown in Figure 2A, ACE and CAF cannot be quanti ied with direct absorbance data because the zeroorder spectrum of the mix solution shows an overlapped spectrum. The zero-crossing points to quantify ACE and CAF were identi ied at 273.0 and 216.5 nm, respectively. At these wavelengths, all analytical   signals were proportional to drugs concentrations.

Linearity, precision, and accuracy
Linear regression equations to test linearity and accuracy of ACE and CAF are shown in Figure 3.
To test linearity all linear regressions were significant (p<0.05). CI 95% for intercepts were: -0.019 to 0.0018 for ACE and -0.0046 to 0.023 for CAF. Considering both drugs the CV ranged from 0.39 to 2.53%. To test accuracy CI 95% for intercepts and slopes were: -3.82 to 3.70 and 0.82 to 1.20 for ACE and -0.52 to 0.38 and 0.87 to 1.17 for CAF. Considering these results, the proposed UV derivative method was a good alternative to determine the in vitro dissolution performance of ACE and CAF in ixed-dose combination formulations.

Uniformity of dosage units and assay
Both commercial formulations met the uniformity of dosage units and assay tests. The percent of ACE and CAF content ranged from 85 to 115% and the assay test was between 90 and 110%. Results are shown in Table 2.

Dissolution pro iles
Dissolution pro iles of ACE and CAF obtained with USP Apparatuses 2 and 4 are shown in Figure 4.
To compare dissolution pro iles (reference vs. generic formulation) model-independent parameters were calculated. Percent of drug dissolved at 60 min (Q 60 ), MDT, DE, and f 2 similarity factor are shown in Table 3. Considering f 2 factor, only dissolution pro iles of ACE were similar (f 2 >50). Some other comparisons match this result. All comparisons of CAF, in USP Apparatus 4, shown  signi icant differences (p<0.05). On the other hand, the in vitro dissolution performance of ACE and CAF was slower with the low-through cell apparatus than the USP Apparatus 2. This kind of behavior has been reported by some authors due to hydrodynamic environment that USP Apparatus 4 generates over the dosage form (Looney, 1997;Brown, 2005;Medina et al., 2014).
To compare dissolution pro iles with a modeldependent approach and after the adjustment of dissolution data to Hyperbola model the t 50% values of ACE and CAF were calculated. Results are shown in Table 4. With these data, similar dissolution pro iles of both drugs were found only with the USP Apparatus 4 (p>0.05).
For a complete model-dependent comparison scheme dissolution data of ACE and CAF were adjusted to common equations. Weibull, logistic, Gompertz, and Probit equations were used. Results are shown in Table 5.
As can be seen in Table 5 and considering the criteria to choose the best-it model, only dissolution performance of ACE in USP Apparatus 2 and CAF in USP Apparatus 4 were well described by the same equation (Probit model). To compare dissolution pro iles of ACE and CAF, under previously described conditions, the t 50% values derived from Probit model were calculated. Results are shown in Table 6. Signi icant differences were found only with ACE data (p<0.05).
Results do not match between comparison approaches used. Due to diversity of the results obtained, it can be concluded that the dissolution pro iles of ACE and CAF between reference and generic formulation are not similar and the drugs do not show equivalence in their in vitro release performance.
Some authors have studied the UV identi ication of ACE/CAF in granules (Szumilo et al., 2019), and in commercial tablets (Dinç, 1999; Vichare et al., . These methods used simultaneous equations to quantify both drugs without mutual interference. In granules, more than 80% of ACE and CAF were released in less than 10 min (Szumilo et al., 2019) and both analytical methods reported for commercial tablets can be used for quality control purposes. Especially in dissolution studies, controlled release of CAF tablets has been reported by some authors (Franek et al., 2014;Tan et al., 2019) and more than 80% of drug dissolved was found between 20-24 h. On the other hand, more than 80% of CAF at 20 min was found in immediaterelease formulations with a binary mixture of drugs and HPLC determination (Williams et al., 2010) as well as with a ternary mixture (Liu et al., 1999). Both dissolution methods used the USP Apparatus 1 at 100 rpm. In this work, an easier and faster procedure with derivative spectrophotometry and

Figure 3: A and B) Standard calibration curves (n=2). C and D) Synthetic mixtures (n=3)
zero-crossing determinations is proposed and similar results were obtained.
Linearity, precision, and accuracy were evaluated with good results. Considering these data, it is possible state that UV derivative method allows the study of the in vitro dissolution performance of ACE and CAF in ixed-dose combination formulations and signi icant differences between dissolution pro iles of generic and reference were found. There are many potential factors that can explain the difference between the branded and their generic counterparts. Those include the manufacturer, apparatus type, surface area of a drug, surfactants, storage, dosage form and the level and type of excipients (Ameri et al., 2012).
Of icial dissolution test for ACE/CAF tablets describes the use of USP Apparatus 2 at 100 rpm and 900 ml of water as dissolution medium (Q>75% at 60 min) (United States Pharmacopeial Convention, 2019). In this comparative dissolution study, we use the USP Apparatus 2 at 75 rpm and the low-through cell apparatus with a controlled pH dissolution medium (0.1 M phosphate buffer pH 7.4) with the aim of evaluate the commercial formulations under a hydrodynamic environment more similar to the gastrointestinal tract. Using our conditions, ACE and CAF reached more than 75% of drug dissolved at 60 min excepting CAF in USP Apparatus 4 (74.20%). Several authors have reported that the proper medium and rotational speed of the paddle is important in assuring that the dissolution test is useful and discriminatory (Shah et al., 1992). Moreover, the maximum luid velocities in the low-through cell apparatus at standard operating conditions are expected to be considerably lower than those found in the paddle or basket apparatuses (D' Arcy et al., 2010).
Although hydrodynamic environments of USP Apparatus 2 and 4 are different, dissolution pro iles comparison helps us to identify if there is an equivalence between dissolution USP Apparatuses. Under certain dissolution conditions, propranolol-HCl (a high solubility drug) has been shown equivalence in its in vitro release performance between USP basket apparatus and low-through cell (Medina-Lopez et al., 2019). On the other hand, the modelindependent parameters MDT and DE have been suggested as suitable parameters to compare dissolution pro iles (Podczeck, 1993;Anderson et al., 1998) and to establish an in vitro/in vivo correlation (Cardot et al., 2007).
Several methods are available to elucidate dissolution data as a function of time, but its dependence on dosage form characteristics can best be deduced by using generic equations which mathematically translates the dissolution curves in the function of other parameters related to delivery device (Lokhandwala et al., 2013). Mathematical models describe the dissolution curves as a function of only a few parameters that can be statistically compared (Adams et al., 2002).
While USP Apparatuses 1 and 2 are currently the most popular methods to study the in vitro dissolution performance of drugs, both methods are operated under closed inite sink conditions and can-not mimic the environment of the gastrointestinal tract (Gao, 2009). The low-through cell apparatus has the advantage of generating a hydrodynamic environment similar to that found inside the digestive system (Looney, 1997). It is important to perform a post-marketing monitoring to ensure safety and ef icacy of ACE/CAF ixed-dose combination formulations with all available analytical methodologies. ACE is considered a hepatotoxic drug and it is easily accessible in several formulations as an overthe-counter medication (Yoon et al., 2016). There are many drug products combined with CAF to enhance the analgesic properties of AINEs and dissolution pro iles comparison of AINEs/CAF generic formulations are scarce.

CONCLUSION
The proposed UV derivative method was applied for determination of ACE and CAF in ixed-dose combination formulations. The method was useful for determination of in vitro dissolution performance of both drugs in reference and generic formulations. It is important to carry out bioequivalence studies with ACE/CAF tablets to relate dissolution data with its in vivo behavior. It is essential to maintain a postmarketing evaluation of formulations with analgesic drugs mixed with CAF to offer the population high quality medicines. More research is needed in this ield.

Funding Support
The authors declare that they have no funding support for this study.