HPLC Helps Asthma Patients Breathe Easierby Dr. Sudesh Bhure, Dr. Nandita Shetgiri, Dr. Sandeep Patil, Dr. Sushama Ambadekar, Analytical Chemistry Division, The Institute of Science, Mumbai, India
Salmeterol xinafoate-fluticasone propionate is an aerosol combination inhaler used in the management of asthma and chronic obstructive pulmonary disease (COPD). Fluticasone, a corticosteroid is the anti-inflammatory component of the combination, while salmeterol treats constriction of the airways. Together, they relieve the symptoms of coughing, wheezing and shortness of breath better than either fluticasone or salmeterol taken on its own.
Other therapies administered in asthma include Salbutamol or Beclomethasone inhalers. These drugs are administered individually or in combination with other active agents. Salmeterol is a long acting beta-adrenoceptor agonist (LABA), usually only prescribed for severe persistent asthma following previous treatment with a short-acting beta agonist such as salbutamol and is prescribed concurrently with a corticosteroid, such as beclomethasone. The primary noticeable difference of salmeterol to salbutamol is that the duration of action lasts approximately 12 hours in comparison with 4 to 6 hours of salbutamol. Inhaled salmeterol works like other beta 2-agonists, causing bronchodialation by relaxing the smooth muscle in the airway so as to treat the exacerbation of asthma.
There are several process related impurities/related substances associated with the manufacture of salmeterol xinafoate and fluticasone propionate drug substance. Different process related impurities are observed with various synthetic routes and/or manufacturing processes. Nine of the related substances studied here are: fluticasone propionate acid propionate, fluticasone propionate dimer, fluticasone propionate acetate, salmeterol impurity H, futicasone propionate S-methyl Impurity, salmeterol impurity G, fluticasone propionate Oxo Impurity, fluticasone propionate chloro impurity and fluticasone propionate iodo impurity.
Very few HPLC methods have been reported for the analysis of salmeterol, its related substances and fluticasone propionate and its related substances respectively. However, no HPLC method has been reported for simultaneous determination of salmeterol xinafoate-fluticasone propionate and their nine related substances in combination inhaler. Also, all the reported methods have made use of conventional HPLC columns with particle size 5 µm and dimensions of 250-mm length and 4.6 internal diameters. The main drawback of these reported methods is longer run time. The developed method makes use of a 1.8-µm rapid resolution high throughput (RRHT) column. The smaller particle size has provided better efficiency whereas the smaller length has facilitated reduced analysis time and back pressure.
The present analytical work presented in this article is a simple isocratic fast HPLC method with ultra violet detection for the determination of salmeterol xinafoate-fluticasone propionate and their nine related substances in combination inhaler.
Materials
Salmeterol xinafoate-fluticasone propionate and its related substances were provided by CIPLA Ltd. Mumbai, India. Disodium hydrogen phosphate (AR grade), triethylamine (AR grade), orthophosphoric acid (AR grade) was purchased from s. d. fine chemicals Ltd. (Mumbai, India). Acetonitrile (HPLC grade), Methanol (HPLC grade) was purchased from Merck (Mumbai, India). In-house purified water (USP) was used.
The HPLC system used was Agilent 1100 Quaternary gradient system equipped with a PDA detector (HP 1100 Series column thermostat, HP 1100 Series pump, HP 1100 Series autosampler with thermostat and Agilent Chemstation Software – Version B.03.02). The Agilent SB C18 column (5 cm × 4.6 mm, 1.8 µm) was obtained from Agilent (USA).
Preparations and chromatography
Buffer
A 0.05-M Na2HPO4 buffer was prepared by transferring 7.098 g of Na2HPO4 to a 1000-mL volumetric flask and dissolving in 990 mL of water (purified, USP). 1 mL of triethanolanine was added to this solution. The pH was adjusted to 2.5 with 10 percent orthophosphoric acid, and the resulting solution was diluted to 1000 mL with water and mixed.
Mobile phase
A 500-mL aliquot of buffer solution was mixed with 350-mL acetonitrile and 150-mL methanol. Phase was filtered using a 0.2-µm filter under vacuum to degas.
Standard solutions
Preparation of Standard Stock Solution (A):
Accurately weighed about 12.0 mg of salmeterol xinafoate WS and transferred into 100-mL volumetric flask. To this, a sufficient amount of diluents was added and mixed, sonicated to dissolve, and cooled. The resulting solution was diluted to volume with diluent and mixed.
Preparation of Standard Stock Solution (B):
Accurately weighed about 12.0 mg of fluticasone propionate WS and transferred into 100-mL volumetric flask. To this sufficient amount of diluents was added and mixed. Sonicated to dissolve and cooled. The resulting solution was diluted to volume with diluent and mixed.
Standard Low load preparation A:
Transferred 1 mL of standard Stock Solution A, 5 mL of Standard stock Solution B and 8 mL of each impurity (Imp) stock solution to 200-mL volumetric flask. Diluted to volume with diluent and mixed.
Resolution solution
Impurity Stock Solution:
Researchers accurately weighed about 5.0 mg of fluticasone acid propionate, fluticasone acetate, fluticasone dimer, salmeterol imp h, s-methyl fluticasone propionate imp, salmeterol imp g, oxo imp, chloro imp and iodo imp into individual 50-mL volumetric flasks. To this, sufficient amount of diluent was added and mixed. Content was sonicated to dissolve and cooled. Resultant solution was diluted to volume with diluent and mixed.
System Suitability Resolution Solution:
Researchers accurately weighed about 15.0 mg of fluticasone propionate WS and 5.5 mg of EPCRS mixture containing salmeterol xinafoate, Impurity E and Impurity G into 25-mL volumetric flask. Added sufficient amount of diluent and mixed. Sonicated the contents to dissolve and added 1 mL of each imp stock solution in same 25-mL flask. Resultant solution was diluted to volume with diluent and mixed.
Sample preparation
The pressurized container was removed from the actuator and all labels and markings were removed, which may be present on the container with methanol. The container was dried, replaced in its actuator and shaken for 5 seconds. It was discharged once to waste, and waiting not less than 2 to 3 seconds, it was shaken for 5 seconds and discharged again to waste. The procedure was repeated for three further actuations. The pressurized container was removed from its actuator; both the valve stem (internally and externally) and the valve ferrule was cleaned by washing with diluent. The complete valve assembly was dried using an air line fitted with an appropriate jet to ensure that all solvent was removed from the inside of the valve stem.
Researchers placed a stainless-steel holder with three legs and a central indentation with a hole (1.5-mm diameter, and about 10-mm height) tapered in a downward direction in a 50-mL beaker. The pressurized container was shaken for about 30 seconds and placed it inverted in a beaker.
Next, 120 sprays were discharged in an empty beaker. The pressurized sample container was shaken between each actuation of valve without removing the pressurized container from its inverted position in the beaker. The content was dissolved into the small amount of diluents and transferred to a 50-mL volumetric flask. Researchers added 30-mL diluent, mixed well and diluted to volume with diluent.
Placebo
A placebo (formulation matrix) was prepared as per the same procedure as that of sample using placebo container.
Chromatographic conditions
Mobile phase flow rate: 1 mL/min; column temperature: 35 C; detection: ultraviolet, 225 nm; injection volume: 20 µL; run time: about 30 min.
System suitability
The system was deemed suitable if the following acceptance criteria were satisfied: The relative standard deviation (RSD) of the peak area responses was not more than 5%; the tailing factor in the resolution solution was not more than 2%; and the resolution between the closely eluting peaks was not less than 1.5.
Limit of detection (LOD) and limit of quantitation (LOQ)
Solutions of salmeterol xinafoate-fluticasone propionate and nine of its related substances were prepared in duplicate from independently prepared stock solutions. Each of the prepared solution was chromatographed. The signal-to-noise ratios for salmeterol xinafoate-fluticasone propionate and nine of its related substances were calculated. The LOD was evaluated as the concentration, which produced a peak with a signal-to-noise ratio of about three. The LOQ was evaluated as the concentration that produced a peak with a signal-to-noise ratio of about 10.
Specificity
Chromatographic profiles
Solutions of salmeterol xinafoate–fluticasone propionate and nine of its related substances were individually prepared and chromatographed. Retention times and relative retention times were determined to evaluate the potential co-elution or interference to the determination of salmeterol and/or the related substances.
Forced-degradation studies
Solutions of salmeterol xinafoate-fluticasone propionate formulation and nine of its related substances were stressed with acidic, basic, oxidative, reductive, thermal and photolytic conditions. Details are presented in Table 1. Prior to analysis, the acid stressed samples were neutralized with base and the base stressed samples were neutralized with acid. The forced degraded samples were analyzed using the Agilent 1100 Quaternary gradient system equipped with PDA detector. A “marker solution” containing salmeterol xinafoate–fluticasone propionate and nine of its related substances was injected within the HPLC run to aid in identification of the degradation products.
Validation studies
Linearity
Solutions of Salmeterol xinafoate–fluticasone propionate and nine related substances were prepared individually in the levels viz. LOQ, 50, 75, 100, 125 and 150 percent. 5 µL of each of these solutions were injected into the chromatographic system under the chromatographic conditions and the peak area was recorded. This experiment was repeated six times for the extreme levels and twice for the range between the extreme levels and the mean, standard deviation and coefficient of variation for each level was calculated.
Accuracy/recovery
Accuracy levels were prepared (from LOQ, 50-percent limit level concentration, 100-percent limit level concentration and 150-percent limit level concentration). Each level was injected in triplicate and the percentage recovery was calculated for each level separately.
Precision
Intra-assay precision or repeatability was carried out by injecting a minimum of six analyte solutions. To prepare each analyte solution, 2 mL of individual stock solution of impurities was diluted to 50 mL with diluent to obtain the concentration of 4 ug/mL. Six replicate assay determination of sample was done and relative standard deviation (RSD) was calculated.
Intermediate precision (also known as ruggedness) was carried out using the same method for repeatability study but with different analysts on different days and on different systems. By injecting six replicate assay determination, the sample is done and RSD is calculated.
Robustness
The robustness of the HPLC system was demonstrated by studying the effects of changes in the HPLC system parameters using a resolution solution. The HPLC parameters varied were column temperature (33 and 37 C) and flow rate (0.9 and 1.1 mL).
Stability of analyte solutions
The stability of the analyte solution was evaluated at room temperature up to 24 hours. Fresh solutions of concentration 4 µg/mL were prepared. The freshly prepared solutions were injected and then kept at room temperature. These samples were analyzed initially (0 hours) and then after 2, 4, 8, 12, 16, 20 and 24 hours.
Results and discussion
Various mobile phases and columns were used to arrive at a method that achieved an optimal separation for all the components. The chromatographic method described here separates all the nine related substances and drug substance salmeterol xinafoate–fluticasone propionate.
System suitability
The typical system suitability requirements were met. The tailing factor for the salmeterol peak was 1.28 (criteria NMT 2.0). The data shows that tailing factor throughout the study was consistently below 2.0.
Limit of detection (LOD) and limit of quantitation (LOQ)
For determining LOD and LOQ the standard solution of the lower concentration of salmeterol xinafoate-fluticasone propionate and its nine related substances were injected. The signal-to-noise ratio for the response were calculated. The results for limit of detection (LOD) are tabulated in Table 2 and are well within the limit i.e., signal-to-noise ratio is between 3 to 10. The results of limit of quantitation (LOQ) are also tabulated in Table 2, which are well within limit i.e., signal-to-noise ratio is above 10.
Specificity
Chromatographic profiles
The specificity of the method was determined by individually chromatographing salmeterol xinafoate–fluticasone propionate and nine of its related substances, Placebo and blank. The chromatograms show that the method is specific and no interferences from the placebo, blank or related substances was observed for the determination of salmeterol or the individual related substances.
Forced degradation studies
Similar stress conditions were applied to both the drug substance and the drug product solutions. The stressed sample of the drug substance showed up to 60-percent degradation (Table 1), while the drug product solutions showed very little or no degradation. The degradation products were well resolved from the drug substance and drug products, as identified by comparing with the marker chromatogram. Peak purity of the salmeterol xinafoate, fluticasone propionate and their nine related substances were found to be within limits.
Validation studies
Linearity
The method was found to be linear with correlation coefficients (R2) ranging between 0.9991 and 1.0000, with slopes near unity and %Y intercepts below 3.00 is within limit.
Accuracy/recovery
The recovery results obtained indicate that the method is accurate for determination of the salmeterol xinafoate–fluticasone propionate and nine of its related substances. The recoveries for the related substances at LOQ level ranged between 90 and 113 percent. The RSD ranged between 0.34 and 3.74 percent. The recoveries determined by the % area approach were well in agreement with the acceptance criteria set for the accuracy/recovery study.
Precision
Method precision: Batch of salmeterol xinafoate-fluticasone propionate combination inhaler was analyzed six times. %RSD were found to be well within the limit set for precision.
Intermediate precision: Batch of salmeterol xinafoate-fluticasone propionate combination inhaler was reanalyzed by another analyst on another system for six times. Results were calculated. The results of intermediate precision study along with repeatability study were compared and found to be well within the limits set for the intermediate precision study.
Robustness
The data obtained from the deliberate variations of the HPLC parameters shows that the variations did not significantly affect the system suitability requirements. The observed responses to the parameter changes were as expected. The results of robustness study along with precision study were compared and found within the limits for flow change and column oven temperature set for the robustness study. This shows that the method is robust for flow and column oven temperature.
Stability of analyte solutions
The results of initial analysis and the results of analysis after preservation for %RSD was compared and found to be well within the set limit for solution stability study up to 24 hours. As the results of initial analysis and analysis after preservation up to 24 hours are comparable, the solution was stable up to 24 hours.
Conclusion
The developed method is novel, fast, specific, and rugged for the simultaneous determination of salmeterol xinafoate-fluticasone propionate and its nine related substances in combination inhalers. Moreover, the total run time of the developed method is 66 percent less as compared to the reported method, resulting in cost reduction of analysis (solvent, manpower, electricity, instrument consumables etc.), time savings and improvement of overall lab productivity.
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