In vitro Evaluation of Trimethoprim and Sulfamethoxazole from Fixed-Dose Combination Generic Drugs using Spectrophotometry: Comparison of Flow-Through Cell and USP Paddle Methods

Purpose: To develop a first-order derivative spectrophotometric method for the determination of trimethoprim (TMP) and sulfamethoxazole (SMX) from fixed-dose combination generic products using a flow-through cell technique. Methods: Absorbance measurement was achieved at 247.8 and 257.9 nm for trimethoprim and sulfamethoxazole, respectively. USP Apparatus 4 with 22.6 mm cells, laminar flow at 16 ml/min, and 0.1 N HCl at 37 °C as dissolution medium, were used. Dissolution profiles were compared with model-dependent and independent methods. Results: All the products met the pharmacopeial dissolution criterion (Q ≥ 70 %, at 60 min), except SMX in two products (SC 400 mg and SB1 800 mg) using the flow-through cell (53.62 and 49.74 % dissolved, respectively). Using both USP apparatuses, significant differences in mean dissolution time and dissolution efficiency values were found (p < 0.05). All products were in line with Weibull’s kinetics and significant differences in derived parameters (Td) values were found (p < 0.05). Conclusion: Determination of TMP and SMX by derivative spectrophotometry can easily be employed for dissolution studies using the flow-through cell technique. However, it would be necessary to determine correlation with in-vivo test results in order to assure safe interchangeability.


INTRODUCTION
Recently, several authors have been worried about safe interchangeability between branded products and its generic counterpart or even among different generic products.For several drugs, interesting and different results have been reported [1,2].Importance of in-vitro dissolution test to guarantee a best quality in generic medications is widely discussed by regulatory organisms [3].According to FDA and WHO guidelines, some generic drugs can be registered on the basis of only in-vitro data (dissolution test) without testing their in-vivo performance [4].In Mexico, as in other parts of the world, trimethoprim-sulfamethoxazol (TMP-SMX) immediate-release oral fixed-dosage forms are marketed as generic drugs.The combination is prepared in different formulations but tablets are the most commonly used, mainly for the advantages of patient management and intake of a solid dosage form.TMP-SMX inhibits bacterial synthesis of tetrahydrofolic acid, the physiologically active form of folic acid and a necessary cofactor in the synthesis of thymidine, purines and bacterial DNA.The fixed 1:5 formulation of TMP-SMX is indicated primarily for treating genitourinary, gastrointestinal, and respiratory tract infections as well as skin-associated infections and HIVinfected patients [5].Development of bacterial resistance and adverse reactions are well documented [6].
According to Biopharmaceutical Classification System, TMP and SMX are classified as Class II drugs [7].Due to their low aqueous solubility, dissolution rate is the rate-limiting step for absorption.TMP and SMX are well absorbed after oral administration however; TMP is absorbed more rapidly than SMX and is more widely distributed throughout the body.Because of this unequal distribution, a widely range of concentrations is achieved in different tissues and body fluids [6].Furthermore, in-vitro dissolution data offer the best method to predict in-vivo performance formulation.In this regard, some authors have been documented differences in in-vitro release characteristics of TMP-SMX commercial products [8].
Spectrophotometric approaches for simultaneous analysis of binary and ternary mixtures in commercial tablets were previously reported [9,10].Studies are often focused on in-vitro dissolution profiles of TMP-SMX without previous extraction steps and interference of matrix effect; however, alkaline or a combination of alkaline/methanolic solutions are usually used in those studies but pharmacopeial dissolution method is carried out in acidic medium (0.1 N HCl).Other kinds of solutions are not the natural environment where drugs will be dissolved within the first minutes after tablets intake.Dissolution profiles of TMP-SMX commercial products with derivative spectrophotometry were also reported.The aid of some devices and continuous-flow methodology knowing as multi-commutation is included [11].For pharmaceutical analysis of TMP-SMX brand products, automated dissolution systems fitted with an integrated multicomponent detector was reported [8].Determination of multicomponent dissolution profiles of TMP-SMX pharmaceutical products by in-situ fiber-optic UV measurements was also described [12].For current pharmaceutical laboratories these equipments are not easily available and routine dissolution profiles comparisons with binary mixtures is difficult to carry out.
For the evaluation of dissolution profiles of TMP-SMX tablets, the United States Pharmacopeia (USP) [13] specifies the use of USP paddle method at 75 rpm with 900 ml of 0.1 N HCl as dissolution medium and not less than 70 % (Q) of TMP-SMX is dissolved in 60 min.HPLC analysis for drugs quantification is recommended.An alternative to evaluate in-vitro drug release is the flow-through cell system (USP Apparatus 4).Its advantages over the conventional basket and paddle methods (USP Apparatus 1 and 2, respectively) are widely demonstrated, especially for the dissolution of poorly soluble drugs [14,15].The USP Apparatus 4 best simulates the hydrodynamic conditions that are found in the gastrointestinal tract.Therefore, it is important to investigate the applicability of the flow-through cell system on the assessment of TMP-SMX dissolution profiles in order to ensure the adequate biopharmaceutical evaluation of fixeddose combination generic drugs.
The aim of this study was to apply a first-order derivative spectrophotometric method, especially developed for dissolution studies (USP paddle method), in the determination of dissolution profiles of TMP and SMX from fixed-dose combination generic drugs obtained with the flow-through cell system.Results were compared with data obtained with the pharmacopeial method, USP Apparatus 2.

EXPERIMENTAL Products and standard solutions
Seven TMP-SMX immediate-release commercial products were used.Different letter was assigned to each one (A, B and C for 80 mg of TMP and 400 mg of SMX tablets) and (A1 and B1 for 160 mg of TMP and 800 mg of SMX tablets).Dissolution profiles of generic drugs were compared to dissolution profiles of the Mexican reference products (R and R1) Bactrim® and Bactrim® F (Productos Roche, SA de CV, Mexico).Hydrochloric acid and methanol analytical grade were purchased from JT Baker-Mexico.TMP and SMX standards were purchased from Sigma-Aldrich Co. (St. Louis MO, USA).All samples were filtered through 0.45 µm nitrocellulose filters (Millipore®, Ireland).
Standard solutions of both drugs were separately prepared by serial dilutions of the stock solutions of TMP (0.2 mg/ml) and SMX (1 mg/ml) in 0.1 N HCl to achieve the concentrations of 10-50 µg/ml of TMP and 250-350 µg/ml of SMX in the same medium.

Content uniformity and assay
Content uniformity and assay tests were performed with all products, according the procedures described in the USP [13].

Analytical method validation
The proposed analytical method was validated according to the International Conference on Harmonization (ICH) guidelines [16].The system linearity, accuracy and precision were analyzed.

Pharmacopeial dissolution method (USP Apparatus 2)
TMP-SMX dissolution profiles were carried out according the procedures described in the USP [13].An USP paddle apparatus (Sotax AT-7 Smart, Switzerland) with a piston pump (Sotax CY7-50, Switzerland) was used.Tablets were added on 900 ml of 0.1 N HCl at 37.0 ± 0.5 ºC (n = 6).Rotational speed of 75 rpm was tested.10 ml of filtered samples were withdrawn at 15, 20, 30, 45, and 60 min and replaced with an equal volume of fresh dissolution medium to maintain a constant total volume.

Flow-through cell method (USP Apparatus 4)
TMP-SMX dissolution profiles were obtained with an automated flow-through cell system, USP Apparatus 4 (Sotax CE6, Sotax AG, Switzerland) with 22.6 mm cells (i.d.) and a piston pump (Sotax CY7−50, Sotax AG, Switzerland).In all experiments, laminar flow (with a bed of 6 g of glass beads) at 37.0 ± 0.5 °C was used.The degassed 0.1 N HCl was used as the dissolution medium, at a flow rate of 16 ml/min and an open system was used.Dissolution samples were taken at 15, 20, 30, 45 and 60 min (n = 6).

First-order derivative spectrophotometric analysis
Simultaneous determination of TMP-SMX was carried out with a first-order derivative spectroscopic method previously developed in our laboratory [17].A double beam UV/Vis spectrophotometer (Perkin Elmer Lambda 35, Waltham MA, USA) with 0.1 cm quartz cells was utilized.The operating conditions for UV analysis were first-derivative mode with scan speed 240 nm/min, slit width 2.0 nm, and sampling interval 1.0 nm.The amounts of TMP-SMX dissolved in both dissolution apparatuses were determined at 247.8 and 257.9 nm respectively, with reference to standard calibration curves.

Data analysis
TMP-SMX dissolution data of each product were used to calculate model-independent parameters: % dissolved at 60 min (Q), mean dissolution time (MDT) [18] and dissolution efficiency (DE) [19].The values of these parameters from generic drugs were compared with the reference products values by ANOVA followed by Dunnett's or Dunnett's T3 multiple comparisons test as appropriate.Data analysis was carried out using SPSS software (Version 17.0).Differences were considered significant if p < 0.05.
Additionally, in order to evaluate the release kinetics of TMP and SMX from the used products, dissolution data were fitted to different kinetic models: First order, Higuchi, Korsmeyer-

Pharmacopeial tests
All products met the content uniformity and assay tests specified in the USP.The percentages of TMP-SMX on the content uniformity test ranged from 85-115 % and the assay test was between 90 and 110 %, Table 1.

Analytical method validation
The mean regression equation from three standard calibration curves was y = 0.0349x + 0.0124 for TMP and y = -0.0100x+ 0.2756 for SMX.The relative standard deviation (RSD) values of response factor were 2.08 and 1.79 % for TMP and SMX ranges, respectively.Considering dissolution of 80, 100 and 120 % of dose, the regression equation to assess the method linearity was y = 1.0005x -0.0227 for TMP and y = 1.0013x + 0.4559 for SMX (R 2 = 0.998; p < 0.05).The method accuracy was

Dissolution profiles
TMP-SMX dissolution profiles obtained with the flow-through cell system and the USP paddle method are shown in Figure 1.Considering a single point specification (Q ≥ 70 % in 60 min) all products met the pharmacopeial dissolution criterion in both USP apparatuses, excepting SMX in products SB (400 mg) and SB1 (800 mg) using USP Apparatus 4 (53.62 and 49.74 % dissolved, respectively).TMP-SMX dissolution test using the USP paddle method did not differentiate between the dissolution profiles; based on the pharmacopeial specifications all products tested reached the Q value.

Model-independent comparisons
MDT and DE mean values ± standard error for products under study in both USP apparatuses are shown in Table 2. Considering modelindependent comparisons significant differences in dissolution profiles of all generic drugs were found.

Model-dependent comparisons
In order to describe the TMP-SMX release kinetics from generic drugs, data were fitted to several kinetics models.Low values of R 2 adjusted and high values of AIC were found with almost all models.The dissolution data of all products in the flow-through cell system and the USP paddle method were best fitted by Weibull's function and the comparison of dissolution profiles was made analyzing the derived parameter (Td) from this function.Significant differences in Td values between generic drugs and the reference products were found (p < 0.05) in both USP apparatuses, Table 3.

DISCUSSION
First-derivative spectroscopic method was successfully applied for TMP-SMX determination together with the flow-through cell apparatus.In USP Apparatus 2 the UV analysis is also adequate because in both apparatuses, TMP and SMX achieved an extent of dissolution of 100 ± 3 % at 60 min from the reference products.In all sampling times the RSD was lower than 3 %.Results in USP Apparatus 4 showed a slower dissolution rate than the one found with the USP paddle method.This behavior can be explained by the hydrodynamic conditions that characterize the flow-through cell, where no agitation mechanisms exists and the dosage form and the drug particles are continuously exposed to a uniform laminar flow, similar to the natural environment of the gastrointestinal tract, causing different in-vitro dissolution pattern [22].Invitro/in-vivo correlation (IVIVC) using the flowthrough cell at flow rates of 8, 16 and 32 ml/min has been previously discussed [23] as well as flow rates of 4, 8 and 16 ml/min are also in the European Pharmacopeia and the USP.
The analytical method validation was done with all products used in the present study however, as an example and in order that method validation is not the main objective of this work, only the reference product R (80/400 mg-dose) data are shown.Moreover, and with reference in the experience of a few countries, the role of generic medicines in healthcare systems and the need to establish and implement generic medicines policies is widely discussed by some authors [28].Suitable in-vitro dissolution studies help to maintain an adequate quality control in formulations that may present potential bioequivalence problems.

CONCLUSION
First-order derivative spectroscopy is a useful technique for the simultaneous determination of TMP and SMX dissolution profiles from fixeddose combination generic drugs using the flowthrough cell method.The USP Apparatus 4 is effective in discriminating in-vitro dissolution characteristics of the different generic products.

Table 2 :
Model-independent parameters: percentage dissolved at 60 min, mean dissolution time (MDT), and dissolution efficiency (DE) of trimethoprim (TMP) and sulfamethoxazole (SMX) from fixed-dose combination generic drugs.Data are mean ± SEM, n = 6: *p < 0.05 Weibull's kinetic model.This model has proven to be useful to describe in-vitro release kinetics of poorly soluble drugs in immediate-release oral dosage forms[14,15].