Simultaneous detection and quantification of bronchodilators in pure form and from in-vitro drug release of a novel combinational formulation

The analytical method was developed and validated for the quantification of salbutamol sulfate (SS) and ipratropium bromide (IPB) in accordance with the International Council for Harmonization guidelines in its pure form. The chromatographic partition was completed utilizing a blend of acetonitrile:phosphate buffer (30:70 v/v) with the pH scale adjusted to 3.0 using o-phosphoric acid at a flow rate of 1 ml/minute in Luna C-18(2)(150 × 4.6 mm i.d., 5 μm) column. The wavelength for detection was fixed at 212 nm. The SS and IPB showed a standard linearity curve in the range of 2–12 μg/ml, with retention time at 2.4 and 3.8 minutes, respectively. The developed method was reported to be specific, linear (r2 ≥ 0.999), precise at intraday and interday levels (% relative standard deviation < 2.0%), accurate (% recovery: 96.02%–103.62%), and robust. The limit of detection and limit of quantification for SS was found to be 0.42 and 1.26 μg/ml, while that of IPB was 0.44 and 1.34 μg/ml, respectively. Additionally, the developed method was effectively applied in quantifying SS and IPB from its pure, commercial, and in-house prepared transdermal system to understanding the in-vitro drug release pattern from patches.


INTRODUCTION
Salbutamol sulfate (SS), chemically known as (RS)-1-(4-hydroxy-3-hydroxy-methyl phenyl)-2-(tert-butylamino)ethanol sulfate, is formulated as a 1:1 sulfate salt. It is the most extensively used sympathomimetic, indicating prominent bronchodilator and less cardiovascular impacts. It mitigates the congestion related to asthma, chronic obstructive pulmonary disease (COPD), and other forms of allergic airways disease (The Merk Index, 2006). The medication is additionally utilized for the anticipation of premature labor and exercise-evoked bronchospasm. Pharmacological action is a result of adenylyl cyclase stimulation, thus increasing the intracellular cyclicadenosine monophosphate levels, promoting relaxation of the smooth muscles of bronchi and the uterus (Springhouse, 2008 ).
The drug is official in European Pharmacopoeia, British Pharmacopoeia, and Indian Pharmacopoeia. Different methods of detection of SS have been accounted for, either alone or in combination with other drugs, using chromatographic analytical techniques, like high-performance liquid chromatography (Walode et al., 2013), liquid chromatography-tandem mass spectrometry (Zhou et al., 2015), gas chromatography (Srichana et al., 1998), and by other nonchromatographic procedures (Basavaiah et al., 2006;Basavaiah et al., 2007). Various other principles for its detection are also reported (Muralidharan and Kumar, 2012) Ipratropium bromide (IPB) is a congener of atropine obtained by treating atropine with isopropyl bromide, chemically described as 8-azoniabicyclo [3.2.1] octane, 3-(3-hydroxy-1-oxo-2-phenylpropoxy)-8-methyl 8-(1-methyl ethyl) bromide (Abdine et al., 2003a). It was designed as an orally active, exceptionally polar drug which inadequately infiltrates the blood-brain barrier, thus preventing central side effects, while the sole significant side effect is dry mouth, at dosages higher than the therapeutic dose. IPB is a white to off-white crystalline substance freely soluble in water and methanol, sparingly soluble in ethanol, and insoluble in lipophilic solvents (Abdine et al., 2003b). The drug is used by inhalation, in the treatment of COPD and allergic rhinitis. It blocks all subtypes of muscarinic receptors, resulting in a decrease in the formation of cyclic guanosine monophosphate (c-GMP) that effectively averts the increase in intracellular concentration of calcium, thereby improving the contractility of smooth muscles and restraining mucus secretion (Mario et al., 2012).
IPB is devoid of structural conjugation (Abdine et al., 2003c); therefore, no analytical methods have been reported by the conventional UV method. The official methods for estimating IPB were the titrimetric method with silver nitrate for the assay of pure substance; BP (1998) used an high performance liquid chromatography (HPLC) method for the assay in pressurized inhalation, while the European, Japanese and American pharmacopoeia described other official methods. Hassan (2000) extended the application of derivative spectroscopy for its estimation. HPLC techniques were reported to quantify IPB in aerosol, liquid for nebulization (Nayak et al., 1992), and its related substances (Simms et al., 1998). Other methods reported are a radioreceptor assay method (Ensing et al., 1988) and a nonaqueous titration method. The detailed spectrophotometric methods depended on the oxidation of IPB with alkaline KMnO 4 and complexation with iodine and bromocresol green (Hassan, 2000).
Not many methods have been accounted for in the simultaneous estimation of SS and IPB by HPLC. Most of the reported methods for their quantification depicted limitations (Jyothi et al., 2012;Kasawar and Farooqui, 2010;Nagaraju and Appaji, 2014;Vankudoth et al., 2013).
These bronchodilators are usually available in the market, either as pressured metered-dose inhalers, dry powder inhalers, soft mist inhalers, or as nebulization solutions. Challenges experienced by incorrect inhaler device usage bringing about befuddled COPD patients prompts suboptimal drug administration, thus rendering poor adherence to medical care (Braido et al., 2016). The alarming rise in mortality and morbidity is caused by the progressive nonreversible disease COPD and the potential drawbacks of the inhaler therapy used in the management of COPD; the economic burden experienced by patients due to hospitalization and exacerbation (Kallaru et al., 2015) happening due to nonadherence of therapy can be resolved by application of the novel drug delivery systems, such as the transdermal drug delivery system which offers numerous advantages, such as increased bioavailability, decreased frequency of dosing, capacity to entrap both hydrophilic and lipophilic compounds, reduced toxicity, and easy termination of medication by removal of the formulation (Ali et al., 2015).
Precise evaluation of the drugs trapped in the matrix is vital, since it is the integral factor that can be related to its therapeutic effectiveness. Hence, we felt the requirement to develop a validated analytical method for the estimation of SS and IPB and to quantify the in-vitro drug release from the in-house manufactured transdermal patch.

Materials
HPLC grade acetonitrile and methanol were obtained from Fisher Scientific (Mumbai, India), IPB (CAS No-18559-94-9) was acquired from Vamsi Laboratories, Sholapur, India, and SS (CAS No-60205-81-4) was gifted by Elegant Drugs, India, and was used as supplied. Evonik Degussa India Pvt. Ltd., Mumbai, was benevolent enough to gift samples of Eudragit Polymer Eudragit RL 100 (RLPO) and Eudragit RSPO. The plasticizer triethyl citrate and mercury were bought from S.D. Fine Chemicals Ltd., Mumbai. Different reagents used were of analytical quality.

Chromatographic conditions
A Luna C18(2) column (150 × 4.6 mm i.d., 5-μm particle size; Phenomenex, CA, USA) fitted with a security guard column (C18 ODS; 4 × 3.0 mm i.d.; Phenomenex, CA, USA) was utilized for the chromatographic separation of SS and IPB. The mobile phase injected at a flow rate of 1 ml/minute involved a blend of acetonitrile:phosphate buffer (pH adjusted to 3.0 ± 0.5 with o-phosphoric acid) in 30:70 v/v ratio. The column temperature was 30°C and the detection wavelength was 212 nm. The mobile phase was filtered and degassed prior to use. The chromatographic run injecting 10-μl volume was stopped at the end of 6 minutes. Before the injection of standards and samples, the column was equilibrized with the mobile phase for 60 minutes. Stock solutions of 1,000 µg/ml of SS and IPB were prepared using the mobile phase and necessary dilutions were made to acquire concentrations of 2-12 µg/ml.

Validation studies
The developed HPLC method was validated in accordance with the guidelines of the International Council for Harmonization (ICH) (International Conference On Harmonization, 2005) for various parameters, such as the system suitability, precision, linearity, accuracy, specificity, robustness, limit of quantification (LOQ) and detection (LOD). System suitability tests were carried out by injecting six replicates of standard SS and IPB solutions and determining the tailing factor, number of theoretical plates, and percentage of relative standard deviation (%RSD) of retention time and peak area. Linearity was measured by using the least squares regression model to assess the slope, y-intercept, and correlation coefficient. The calibration curves were obtained from various SS and IPB concentrations ranging from 2 to 12 µg/ml and were tested in triplicates. Precision was evaluated by determining the repeatability of three different standard solutions for six times on the same day (intraday) and examining the same three standard arrangements three times on three successive days (interday). Accuracy was tested at three diverse concentration levels by spiking the pre-analyzed commercially available samples with known concentrations of SS and IPB and later deciding the %RSD. Specificity was studied by checking the blank patches chromatograms amid in-vitro release to check for potential interference by the excipients added. The LOD and LOQ values were decided numerically based on the standard deviation of the response and the slope. They were expressed as LOQ=3.3 σ÷S and LOQ=10σ÷S. The robustness of the developed reverse phase high performance liquid chromatography (RP-HPLC) method was evaluated by deliberate changes to the optimized chromatographic conditions based on the %RSD values of the system suitability parameters, such as change of ± 0.2 ml/minutes, ± 2%, ±5°C, and ± 2 nm in the flow rate, mobile phase composition, column temperature, and wavelength, respectively.

Fabrication of patches
Four films coded F1, F2, F3, and F4 were prepared according to the composition stated in Table 6, by dissolving the drugs to the previously prepared methanolic polymeric solution comprising plasticizer, sonicated to remove entrapped air bubbles, and the final mixture was made to 10 ml. Drug-loaded polymer solutions were casted onto a flat-bottomed Anumbra petri dish using mercury as a substrate and dried overnight at room temperature (Parhi and Suresh, 2016).

Formulation considerations
The total dose (Eriksen and Robinson, 2006) for oncedaily controlled-release formulation of SS and IPB was calculated using the available pharmacokinetic data as per Robinson Eriksen's equation. The loading dose for incorporation into the formulation was calculated considering the radius 4.4 cm of the Anumbra molds (Parhi and Suresh, 2016).

In-mano evaluation
The prepared films were graded either as transparent or translucent based on the opacity of the film, their texture was given two grades (smooth/rough), and finally, the nature of the film was noted as either flexible or brittle. The patches which could withstand in-mano stress to maintain its structural integrity and the film which could be rolled and folded were termed as tensile and pliable, respectively (Ham et al., 2013). A study of tack properties enabled us to understand the adhesive nature of the film. It was studied a week after the preparation of the transdermal patches, by pressing the thumb lightly on a patch for about 5 seconds and then quickly withdrawing. The folding endurance was measured manually by subjecting a small strip of the film (2 cm × 2 cm) to repeated folds in the same place. The number of times the film was folded without developing visible cracks or breaking was noted (Shailesh et al., 2011).

Surface pH
The surface pH of the patches was determined by Bottenberg et al.'s method and reported in Table 6. The surface pH of a 1-cm 2 patch, previously soaked in 0.5 ml double-distilled water for 1 hour, was noted by bringing a glass electrode close to the surface of the patch and permitting it to equilibrate for 1 minute (Bharkatiya et al., 2010).

Drug content
Patches of 1-cm 2 area, placed in tubes containing 10-ml mobile phase, were shaken with the help of a rotary flask shaker for 24 hours at room temperature, till the complete dissolution of the drugs. After prior filtration, 0.5 ml drug aliquots was pipetted out into vials containing 1.5 ml of mobile phase, and each film formulation was tested in triplicate and the results are expressed as the mean and standard deviation in Table 6 ( Vijaya et al., 2012).

In-vitro drug release studies
A Franz diffusion system having six vertical 12.5-ml capacity receptor cells with an effective diffusion area of 1.77 cm 2 and a PermeGear diffusion system (Perme Gear, Inc. Hellertown, PA) filled with phosphate buffer pH 7.4 were used as the diffusion medium. A magnetic stirrer bar of 10 × 2.5 mm served for the continuously stirring the receptor solution maintained at 37°C ± 0.5°C. The dialysis membrane of pore size 0.45 μ, soaked in phosphate buffer overnight, was mounted between the donor and receptor compartments so that it just touched the receptor fluid's surface. The prepared transdermal film was placed on the dialysis membrane and covered with aluminum foil, 0.5 ml sample aliquots diluted to 2 ml was withdrawn every 2 hours (for 24 hours), and the released drug was quantified by detecting using the HPLC at 212 nm. The drug release rate is estimated and shown in Figure 3 and 4.

Analysis of commercial formulations
The application of the method in estimating drug content from marketed formulation was tested by appropriate dilution of capsules and the results are shown in Table 4. The typical chromatograms of a pure, commercial formulation and in-vitro release pattern are shown in Figure 2.

Absence of conventional method
The concern in evaluating the stated drugs simultaneously by the simple analytical technique UV-visible spectrophotometry was confirmed by the trials of the simultaneous equation method and the derivative spectroscopic method. The low absorbing nature of IPB and almost similar zero-crossing points made their simultaneous estimation tedious. Subsequently, our aim was to build another method of analysis for the estimation of these drugs.

Optimization of chromatographic conditions
The RP-HPLC method was optimized by changing different parameters, such as mobile phase composition, flow rate, detection wavelength, and column oven temperature, with the true objective to acquire sharp and symmetrical peaks with excellent baseline separation. The Luna C18(2) column (150 × 4.6 mm i.d., 5 μm) was used as the stationary phase and the detection wavelength was fixed at 212 nm due to enhanced sensitivity. No drug elution was observed during the trials comprising methanol-water, methanol-acetonitrile, methanol-potassium and dihydrogen phosphate buffer in various proportions, but when methanol is replaced by acetonitrile, peaks with shorter retention time were eluted. Therefore, further trials were carried out by altering the proportions of acetonitrile and buffer. The ACN:PBS (30:70 v/v) ratio was finalized and gave well-resolved peaks with no significant improvement in peak shape by additional trials. Mobile phase was also considered as a diluent to avoid disturbances in the baseline. The temperature was set at 30°C and the flow rate at 1.0 ml/minute, thus resulting in a well-resolved peak with better shape, shorter retention time, and lesser tailing factor.

METHOD VALIDATION
Analytical method validation is carried out to guarantee that the developed method is reliable and adequate for its intended purpose. The proposed method was validated in accordance with the ICH guidelines by reviewing parameters of system suitability, specificity, linearity, accuracy, precision, LOD, LOQ, and robustness.

System suitability
System suitability test guarantees that the set chromatographic conditions ensure proper separation, and the parameters are abridged and presented in Table 1. The high number of theoretical plates (n > 2,000) exhibited good column efficiency and the tailing factor values (T<2) indicated good peak symmetry. The %RSD of the retention time and peak area was within 1%, indicating the chromatographic setup's reproducibility and repeatability (%RSD<1). The resolution (Rs) of 4.8 between SS and IPB indicated a high degree of peak separation (Rs>2.0). All tested parameters for further validation and sample analysis were within acceptable limits, suggesting the suitability of the chromatographic arrangement.

Specificity
The retention times of SS and IPB were 2.4 and 3.8 minutes, respectively. No peaks were eluted when the mobile phase alone or blank patches were exposed to similar experimental conditions as that of drug-loaded films, while the drug-loaded patches and the commercial formulations showed distinct peaks as that of pure drugs, thus indicating no interference from the formulation ingredients. Chromatograms depicting specificity is shown in Figure 1.

Linearity, detection, and quantification limits
The calibration curves of SS and IPB were linear over the tested concentration range of 2-12 μg/ml and the coefficient of correlation for both bronchodilators was higher than 0.999, proposing an excellent correlation and good linearity for the method developed is presented in Table 1. The LOD values were found to be 0.42 µg/ml for SS and 0.44 µg/ml for IPB, while the LOQ values were found to be 1.26 µg/ml for SS and 1.34 µg/ml for IPB, indicating a sufficiently sensitive method to determine the drug even from low permeable matrix systems.

Precision
Intraday and interday precision results are expressed in %RSD in Table 2 and in both cases were found to be below 2%, demonstrating that the developed method is precise. Table 3 summarizes the mean %recoveries, indicating the low variability and closeness between experimental and true values. Table 5 depicts the robustness data with %RSD values (<1) and system suitability parameters (theoretical plate, n > 2,000; tailing factor, T<2). The parameter tested was found to be satisfactory, and hence confirms that the developed method is robust.

Consideration of formulation parameters
SS of 5 mg and IPB of 1 mg per patch were found as the optimum dose. Our aim was to prepare an 8-cm 2 patch while the area of the anumbra plate was found to be 60.7904 cm 2 . Hence, the amount of the drug required was approximately 40 mg of SS and 8 mg of IPB.

In-mano evaluation
All formulations appeared to be as transparent, flexible, tensile, pliable, and have a smooth texture. All the stated characteristics were more vivid with the increasing content of RL. Hence, the developed films are said to possess the prerequisites for an optimized patch. The tackiness of the films was in the following order F1>F2>F3>F4. This pattern can be correlated with the direct proportionality between tackiness and RS content. The effect of the plasticizer, polymer, and solvent is ruled out, since these factors were unaltered in these formulations. The films exhibited no signs of folds even after 100 folds, specifying the ability of the films to be unaffected when it experiences the natural fold of the skin.

Surface pH
The surface pH of all the formulations is presented in Table 6, and the results indicate the compatibility of the prepared patch with the skin.

Drug content
Drug content analysis was carried out for all the prepared transdermal systems and the results displayed in Table 6 show good uniformity and a higher degree of reproducibility with a low standard deviation. The drug content from the marketed formulation is presented in Table 4.

In-vitro drug release studies
The developed RP-HPLC method was also extended to quantify the drug release pattern of the in-house manufactured patches and the in-vitro release profile of SS and IPB from formulations is graphically shown in Figures 3 and 4, respectively.

CONCLUSION
In conclusion, the strategically developed, ICH-validated RP-HPLC method for successful quantification of SS and IPB content in pure and marketed formulation is hereby stated. The validation parameters are in accordance with the ICH guidelines. This method adequately helps to quantify drugs released from in-house manufactured transdermal patches at regular intervals and was found to be more economical, namely factors showing decreased elution time (6 minutes), flow rate (1 ml/minute), and consumption of costly solvents.