INTRODUCTION

Chronic bronchitis and emphysema are the two existing lung diseases in which the airway become narrow and is collectively named as chronic obstructive pulmonary disease (COPD).¹

Essential management approaches are stopping smoking habit, vaccinations, rehabilitation and treatment by using inhalers. The combination of formoterol fumarate (FF) and tiotropium (TIO) is used in targeting various characteristics of COPD as bronchodilation and the inflammations.^1,2

FF dihydrate is a directly acting sympathomimetic with beta-adrenoceptor stimulant activity. FF is prescribed for its long acting beta 2 agonist effect for treating airway obstruction, asthma and COPD.³ The pharmacological effect of beta 2 agonist is to stimulate intracellular adenyl cyclase enzyme that catalyzes the conversion of adenosine triphosphate to cyclic-3’,5’-adenosine monophosphate (cyclic AMP). Increased cyclic AMP levels causes relaxation in the release of immediate hypersensitivity mediators from mast cells. Chemically, it is N-2-hydroxy-5-(1RS)-1-hydroxy-2-(1RS)-2(4methoxyphenyl)1methylethylaminoethyl phenyl formamide(E)-butenedioatedihydrate with molecular formula C₄₂H₅₂N₄O₁₂·2H₂O and molecular weight of 840.92.^1,2

TIO bromide monohydrate is an anticholinergic, antimuscarinic bronchodilator used in the airway obstruction, COPD conditions.^1,2,3 TIO shows its pharmacological effects by inhibiting M3 receptors  in the smooth muscle, which leads to bronchodilation. Chemically it is (1R,2R,4S,5S,7s)-7-(2-hydroxy-2,2-dithiophen-2-ylacetyl)oxy-9,9-dimethyl-3-oxa-9-azoniatricyclo3.3.1.02,4 non-anebromidemonohydrate with molecular formula C₁₉H₂₂BrNO₄S₂·H₂O and molecular weight of 490.40.¹

A complete literature survey reveals that TIO is determined by spectrophotometric method.⁴ TIO in bulk and dry powder inhalation (DPI) form is determined by high performance thin-layer chromatography.⁵ Methods are available to determine TIO and  related substances by high performance liquid chromatography (HPLC).⁶ For the biological estimation of TIO in human plasma; three methods illustrated.^7,8,9 Estimation of FF in various pharmaceutical dosage forms by spectrophotometry with charge transfer complexation technique,^10,11 Q absorbance ratio and solving simultaneous equation,¹² and zero order spectrophotometric method and area under curve technique.¹³ FF also estimated along with other drug moieties by thin layer chromatography densitometry methods.^14,15,16,17 FF also estimated along with other drug moieties in HPLC,^{14,17,18,19,20,21,22,23,24} also in plasma, urine and biological samples.^25,26 TIO has been determined by either FF^27,28,29 or ciclesonide or olodaterol^30,31,32,33 in various dosage forms by HPLC methods, but the  focus was found to be on a single drug compound. In FF the hydrazine hydrate content is determined by gas chromatography-mass spectrometry method.³⁴ Moreover, no related substances analytical method available in any of the pharmacopeias.

To the best of the author’s knowledge, no simple, sensitive and robust related substances analytical method, which focused on both the drug moieties reported till now for the simultaneous evaluation of TIO and FF in DPI dosage form and validated according to International Council for Harmonisation (ICH) guidelines.³⁵ The proposed validated reversed-phase-HPLC method can therefore be applied for simultaneous evaluation of TIO and FF QC testing and stability studies for the determination of related substances. To perform this study Tiomate Transcaps^® DPI manufactured by Lupin ltd. India is used.

MATERIALS AND METHODS

Instrumentation

The Dionex HPLC system consists of dionex ultimate 3,000 UHPLC system equipped with a quaternary gradient pump dionex ultimate 3,000 pumps, dionex ultimate 3,000 auto sampler, dionex ultimate 3,000 column compartment and a dionex ultimate 3,000 UV-Photo Diode Array detector. Separation and quantitation were carried out using a C18 Hypersil BDS column (250 mmx4.6 mm, 5 µm) Chromeleon 7.2 SR5 software used for data acquisition.

Chemicals and reagents

Pharmaceutical respiratory-grade TIO was provided and qualified by Vamsi lab Ltd (India) as such assay was found to be 101.79%. Pharmaceutical-grade FF was provided and qualified by Vamsi lab Ltd (India) as such assay was found to be 100.12%. HPLC grade acetonitrile (Rankem), Milli-Q water (Milli-Q^® CLX 7000), sodium dihydrogen phosphate monohydrate, triethylamine, orthophosphoric acid (Rankem), 0.45 µm Buffer filter (mdi) was used.

Chromatographic conditions

The chromatographic separation utilizes a gradient elution in which buffer consists of 1.38 mg of sodium dihydrogen phosphate monohydrate in 1,000 mL of water, add 2 mL of triethylamine, adjust pH 3.2 with dilute orthophosphoric acid, filter and degas through 0.45 µm filter. Mobile phase A is buffer solution pH 3.2 and mobile phase B is acetonitrile 1.0 mL min^-1 flow rate and BDS Hypersil C18 (250×4.6 mm, 5 µm). Diluent consists of a mixture of buffer pH 3.2 and acetonitrile in the ratio of 70:30 v/v. Analysis was carried out at 30°C column temperature and photodiode-array detection (PDA) detector at wavelength 240 nm for both TIO and FF. The injection volume was 100 µL and run time was 50 min. The Retention time of FF and TIO was found to be at 7.8 and 10.3 min, respectively.

The gradient program is as follows::

Standard preparation

TIO standard stock solution

Standard solutions of TIO were prepared by taking 36-mg TIO separately in each 100 mL volumetric flask, added 70 mL of diluent sonicate to dissolve and make volume with diluent and mix. Further dilute 5 mL of this solution to 100 mL with the diluent.

FF standard stock solution

Standard solutions of FF were prepared by taking 24-mg FF separately in each 50 mL volumetric flask, added 35 mL of diluent sonicate to dissolve and make volume with diluent and mix. Further dilute 1 mL of this solution to 100 mL with the diluent.

Mix standard solution

Pipette out 5 mL of TIO standard stock solution and 10 mL of FF standard stock solution to 100 mL with diluent.

Sample preparation

Tiomate Transcaps^® (Lupin ltd..) preparation, carefully open and collect the sample powder equivalent to 0.72-mg TIO in to 10 mL volumetric flask, added about 7 mL diluent sonicate for 15 minutes with intermediate shaking, cool and dilute to volume with diluent and mix well and filter the solution through 0.45 µm filter by discarding the first few mL of the filtrate and use.

Procedure

Separately inject equal volume of the diluent, placebo solution, standard and sample solutions, record the peak responses. Disregard any peak area due to diluent, FF and placebo solution in the sample solution. Calculate the % of each impurity present in the sample solution by following formulae:

Calculation:

where,

AT: Area of each impurity in the sample solution, As: The following: Area of standard solution 1, Wt. std.: Weight of standard in mg, Wt. spl.: Weight of sample in mg, Avg. Wt: Average weight of net content in mg, L.C.: Label claim in mcg, P: Potency of standard, 392.5: Molecular weight of tiotropium, 490.4: Molecular weight of tiotropium bromide monohydrate.

Analytical method development and optimization

The milli-Q water in different proportions of methanol and acetonitrile tried in both isocratic and gradient elution as well by using various C8 and C18 columns but no proper separations were achieved. Different proportions of potassium and sodium salt buffer (10 mMol to 30 mMol) with methanol and/or acetonitrile were used in various proportions in both isocratic and gradient elution patterns but no proper peak shape, tailing factor and theoretical plates of TIO and FF were observed; also resolution between TIO and FF was not good.

Various ranges of pH were tried from pH 2.5 to pH 6.5 and found that the best results were obtained with sodium dihydrogen phosphate monohydrate buffer pH 3.2 and acetonitrile 1.0 mL min^-1 flow rate and BDS Hypersil C18 (250×4.6 mm, 5 µm). Diluent consists of a mixture of buffer pH 3.2 and acetonitrile in the ratio of 70:30 v/v. Analysis was carried out at 30°C column temperature and PDA detector at a wavelength 240 nm for both TIO and FF. The injection volume was 100 µL and run time was 50 min. The retention time of FF and TIO was found to be at 7.8 and 10.3 min respectively.

Analytical method validation parameters

The comprehensive and systematic method validation was carried out as per ICH guidelines. The analytical method was validated for system suitability, system precision, method precision, intermediate precision, ruggedness, specificity, selectivity, forced degradation, linearity & range, accuracy, limit of detection (LOD) & limit of quantification (LOQ) determination, precision at LOQ level, filter validation, robustness (change in chromatographic conditions) and stability of an analytical solution.

System suitability and system precision were determined by injecting two and six replicate injections of the standard solutions, respectively. The responses of peaks were recorded.

In LOD and LOQ determination, a series of standard preparations of FF and TIO standard over the range starting from 1% to at least 50% of standard concentration was prepared. Plotted linearity graph of average area at each level against the concentration (ppm) and determine the correlation coefficient, slope and intercept of analyte for LOQ determination. The concentrations for LOD & LOQ from linearity study were determined.

Method precision may be defined as the precision of an analytical procedure expressing the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. In method precision six samples were prepared as per the analytical method representing a single batch; % impurities of these samples were determined for both the analytes and the analytical method precision was assessed by the % relative standard deviation (RSD).

Intermediate precision (ruggedness) expresses the ability of an analytical method to remain unaffected and produce reliable results within the laboratory variations such as different days, different equipment, different analysts. Six samples were prepared as per the analytical method representing the same batch used for method precision. % impurities of these samples were determined for both the analytes. The method precision and intermediate precision was assessed by the overall % RSD.

The specificity (selectivity) study is conducted to prove the ability of an analytical method to assess unequivocally the analyte in the presence of components which may be expected to be present in the sample. The diluent, placebo solution, FF dihydrate selectivity solution, TIO selectivity solution, fumaric acid selectivity solution, standard and sample solution were prepared as mentioned in the analytical method, injected and recorded the observations for both TIO and FF.

In forced degradation study, the sample and placebo were exposed under relevant stress conditions such as temperature, oxidation, photolytic, humidity, acid hydrolysis and base hydrolysis. Samples of these stress conditions were analyzed as per the analytical method described. The experiment was performed to achieve 5-30% of degradation in at least one stress condition.

Linearity & range; the linearity of an analytical procedure is its ability within a given range to find test results that are directly proportional to the analyte concentration in the sample solution. TIO and FF standards were prepared in a range of LOQ to 150% of the working standard concentration. Linearity graph of concentration vs. average peak area of the analyte was plotted separately. The correlation co-efficient, slope, and y intercept were evaluated.

The accuracy expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value obtained using the method. The samples for accuracy were prepared as per spiking the TIO and FF standard solution in the placebo at LOQ level, 50%, 100%, and 150% concentration level of standard in triplicate for 50, 100, 150% and six times for LOQ level of working concentration and analysed as per the described method.

For the filter study, the sample solution was prepared as described in the analytical method. The solution was centrifuged at 4,000 rpm for 10 minutes. Decanted supernatant solution was injected as centrifuged sample solution. From the remaining half portion of the solution, filtered the solution through 0.45 µm nylon filter and filled the vials by discarding 0 mL, 2 mL and 5 mL solution. These solutions were injected as a sample solution. The peak responses were recorded for both the analytes for all centrifuged and filtered solution in single sequence.

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in the analytical method parameters and provides an indication of its reliability. In this study, parameters like change in detection wavelength, flow rate, column oven temperature, mobile phase organic composition (acetonitrile) and mobile phase buffer pH were performed and peak responses were recorded for both analytes.

For solution stability, the standard and sample solutions for both FF and TIO were prepared and injected against freshly prepared standard solution on day-0, day-1, day-2, and day-3.

RESULTS AND DISCUSSION

System suitability & system precision

System suitability is demonstrated by preparing duplicate standard solution of TIO and FF and injecting the same. System precision is demonstrated by injecting standard solution of TIO and FF in six replicate injections according to the analytical method described above. For system suitability the similarity factor for both standard solution 1 and standard solution 2 should be between 95.0% to 105.0% for both TIO and FF. For system precision, the similarity factor for six replicate injections of standard solution 1 should be between 95.0% to 105.0% for both TIO and FF. The number of theoretical plates should not be less than 2,000, tailing factor should not be more than 2.0 and capacity factor should be more than 1.0 for both FF and TIO peaks (Table 1, 2).

LOD and LOQ determination

Prepare a series of standard preparations of FF and TIO standard over a range starting from 1% to at least 50% of standard concentrations (Figure 1). A series of low concentrations ranges from 0.007 ppm to 0.365 ppm for TIO and 0.005 ppm to 0.243 ppm for FF has been prepared on the basis of standard response and injected in triplicate injections. The calibration curves were prepared for area vs. concentration for TIO and FF is given below. From these calibration curve slope; intercept and correlation coefficient from the Microsoft Excel along with the STEYX were determined and the LOD & LOQ were calculated as per below formula (Table 3, Figure 2, 3).

For TIO,

LOD        = 3.3 x STEYX/slope

                =3.3 x 0.00241

                =0.008 PPM/p>

Reported value in PPM = NA

LOQ        =10 x STEYX/slope

                = 10 x 0.00241

                = 0.024 PPM

Reported value in PPM = 0.015

From the prediction linearity study statistically calculated LOD and LOQ values are, LOD is 0.008 ppm and LOQ is 0.024 ppm and reported LOQ = 0.015 ppm i.e. 0.02%.

For FF,

LOD        =3.3 x STEYX/slope

                =3.3 x 0.00210

                =0.007 PPM

Reported value in PPM = NA

LOQ        =10 x STEYX/slope

                =10 x 0.00210

                =0.021 PPM

Reported value in PPM = 0.01

From the prediction linearity study statistically calculated LOD and LOQ values are, LOD is 0.007 ppm and LOQ is 0.021 ppm and reported LOQ = 0.01 ppm i.e. 0.02%.

Method precision & intermediate precision (ruggedness)

In method precision, as per the analytical method, six sample preparation were prepared representing a single batch. The intermediate precision or ruggedness was verified by performing precision study as per the analytical method six sample preparation of a single batch sample by different analyst, on different day, using different column and on different instrument. As per ICH guideline Q2 (R1), The % single maximum impurity (above LOQ level), % total impurity, the mean of % single maximum impurity (above LOQ level), and intermediate precision were calculated the % RSD of results of % single maximum impurity (above LOQ level) & % total impurity of six sample preparations should not be more than 15.0 (Table 4).

Specificity (selectivity)

Prepared diluent, placebo solution, FF selectivity solution, TIO selectivity solution, fumaric acid selectivity solution standard and sample solution, as mentioned in analytical method and injected and recorded the observations. The diluent and placebo should not give any interfering peak at the retention time of FF and TIO peaks. The peak purity should pass for the analyte peaks in the standard and sample solution. FF is a fumarate salt prepared from arformoterol, in a chemical reaction for every two molecules of formoterol one molecule of fumaric acid is released. Aim to inject fumaric acid selectivity solution is to identify the retention time of fumaric acid and to confirm that it is not interfering with the retention time of FF and TIO peaks and based on the above observations the method is found to be selective (Table 5, Figure 4a, b, c, d, e, f).

Forced degradation

Forced degradation study is conducted to generate the data for estimating finished drug product stability. The forced degradation study consists of an appropriate solid and solution state stress conditions as per ICH guidelines. Intact capsules were kept at different stress conditions and were withdrawn at the exact time and samples were prepared according to each condition mentioned. The entire runtime was about double the retention times of both FF and TIO peaks. The degradant peaks should be well separated from the FF and TIO peaks also peak purity should pass for the FF and TIO peaks in the degradation samples as shown in (Figure 5a, b, c, d, e, f, g, h). The sample and placebo were degraded in the following manner mentioned in (Table 6).

Linearity & range

The linearity of related substance analytical method for FF and TIO in FF and TIO DPI was performed in standard concentrations over the concentration levels ranging from LOQ to 150% of the standard solution standard concentration for each TIO and FF is considered  100% that is 0.015 ppm to 1.089 ppm for TIO and 0.01 ppm to 0.728 ppm for FF. Linearity graph of concentration vs. average peak area of analytes plotted. The correlation coefficient between concentration (ppm), peak area slope and y intercept evaluated. The correlation coefficient should not be less than 0.999 for both analytes (Table 7, Figure 6, 7).

Accuracy

FF and TIO standards were spiked in placebo at different concentration levels i.e. LOQ level, 50%, 100% and 150% of targeted concentration and analyzed as per method described that is 0.0148 ppm to 1.1129 ppm for TIO and 0.01 ppm to 0.7464 ppm for FF. % recovery obtained at concentration levels LOQ, 50%, 100% and 150% is reported in (Table 8).

At LOQ-level % recovery should be between 80.0% to 120.0% and % RSD of recovery at LOQ level should not more than 15.0 and at 50%, 100%, and 150% level, % recovery should be between 85.0% to 115.0% and % RSD of recovery should not more than 15.0. The result observed are within the acceptance criteria, therefore the method is accurate throughout the selected range.

Filter study

The prepared sample solution and analysed centrifuged and filtered sample solution through nylon filter 0.45 µm in single sequence. The absolute % difference for % single maximum impurity (above LOQ level) and % total impurity between filtered and centrifuged sample solution should not be more than 2.0. hence 0.45 µm nylon membrane filters can be used, and it is recommended to discard the first 5 mL of the sample solution in the routine analysis (Table 9).

Robustness

The % RSD of the area of five replicate standard injections, theoretical plates and tailing factor of TIO peak in each replicate injection were recorded and reported (Table 10).

Solution stability

The standard and sample solutions for FF and TIO were prepared on day 0 of experiment, stored these solutions at room temperature for every time interval up to 3 days and analyzed these solutions on subsequent days. The standard solution was prepared freshly and calculated the assay of analyte in the standard solution and % impurities in the sample solution.

The cumulative % RSD of % assay of the stored standard solution should not be more than 5.0.

The % single maximum impurity (above LOQ level) & % total impurity for samples should comply with the specification limits. The cumulative% RSD of impurity results (above LOQ level) obtained using stored sample solutions should not be more than 5.0.

The solution is considered stable, until the time point where the % RSD of the stored standard and sample solution is not more than 5.0; thus, the solution is stable up to 2 days at room temperature is proved (Table 11).

CONCLUSION

The recommended analytical method for the related substance determination of Tiomate Transcaps^® DPI is simple, robust, selective, specific and precise. It also demonstrates the study of degradation pattern; therefore, can be used for quality control testing, routine analysis and for stability studies.

ACKNOWLEDGMENTS

The authors are grateful to SAVA Healthcare Ltd.; Pune India, for providing the facilities to conduct this work.

Conflict of interest: No conflict of interest was declared by the authors. The authors are solely responsible for the content and writing of this paper.