STUDY OF PRESSURISED METERED DOSE INHALERS FOR THE PURPOSE OF STANDARDIZATION OF QUALITY ATTRIBUTES CHARACTERIZING UNIFORMITY OF DOSING

Aim. The purpose was to provide the rationale of test in regard to uniformity of fine particles dose for pressurised metered dose inhalers (pMDIs). Materials and methods. The pMDIs containing suspensions of salbutamol sulfate (SS) or solutions of beclometasone dipropionate (BD) were studied by laser diffraction and high performance liquid chromatography (HPLC). The particle size distribution of SS, the average dose mass and uniformity of dose mass, the average delivered dose and the uniformity of delivered dose, the average fine particles dose and uniformity of fine particles dose were determined. Apparatus A was used for assessment of fine particles dose. Results. The two analytical procedures for the quantitative determination of SS and BD by HPLC were validated in the ranges with low concentrations of these substances. The 5 medicinal products in pMDI dosage form were studied: 3 preparations were with SS and 2 ones contained BD. It was shown that three products with SS were very similar in regard to particle size distribution in containers and the average values of delivered dose were almost the same, but these products were different in the average dose mass and fine particle dose. According to the research results, the expediency of determining the average dose mass and the tests concerning uniformity of dosing of preparations by dose mass and by fine particle dose was substantiated. It was shown that in the case of pMDI the dosing of solutions of BD was more uniform compared to suspensions of SS. The approaches of leading and other pharmacopoeias concerning uniformity of dosing for pMDIs were critically discussed. The expediency of determination of uniformity of fine particle dose at the stage of pharmaceutical development was substantiated, as the therapeutic effect depends on fine particle dose. Issues concerning standardization pMDIs in regard to uniformity of fine particle dose were discussed. Conclusions. The expediency of standardization and quality control of pMDIs in regard to such attributes as the average dose mass, which characterizes the volume of the metering chamber of the valve as well as the uniformity of the dose mass and the uniformity of fine particle dose, which assure the therapeutic effect of each dose of the product was substantiated


Introduction
The main regulatory and guidance documents, which outline the requirements for the quality of pressurized metered dose inhalers (pMDI), are the European Pharmacopoeia [1], in particular, the general articles "Preparations for inhalation", "Pressurized pharmaceutical preparations" and "2.9.18. Preparations for inhalation: aerodynamic assessment of fine particles" as well as document EMEA/CHMP/QWP/49313/2005 Corr. "Guideline on the Pharmaceutical Quality of Inhalation and Nasal Products" [2]. Аccording to these documents several types of dose can be distinguished for pMDI. The dose is the quantity of the active substance to be administered at one time. The metered dose (MD) is the quantity of an active substance contained in the metering chamber of the valve. The delivered dose (DD) is the quantity of an active substance that is available to the user, exdevice, on a per dose basis; this dose is the part of the metered dose except for that part which has deposited on the actuator. Delivered dose is also the dose delivered from the actuator to the apparatus used to characterize pMDI. [1,[3][4][5][6]. The fine particle dose (FPD) is the part of delivered dose that is consisted from small particles of an active substance (≤ 5 μm) that are capable to penetrate into the lung during inhalation and provide therapeutic effect. The other part of delivered dose contains larger particles of an active substance that deposit in the mouth and throat of the patient and do not provide therapeutic effect [7,8]. The small and larger particles contained in these two parts of delivered dose settle on different stag-es of apparatuses for determination of fine particle dose and particle size distribution [9,10]. Metered dose, delivered dose and fine particle dose are determined on a per one actuation basis.
The metered dose is attributable to the formulation, which is developed taking into account the nominal dose of an active substance and the volume of the metering chamber of the valve. Therefore, the metered dose is very close to the nominal dose stated in the label, and the variability of the MD is due to formulation variability from batch to batch as well as the tolerance on the nominal volume of the metering chamber of valves. Usually metered dose is used for quantitative determination of an active substance. For this purpose it is necessary to remove actuator and discharge 10 deliveries below the surface of the solvent maintaining the pressurised container in the vertical plane and discharging the preparation by actuating the valve [3].
The delivered dose depends on the metered dose and the design of the actuator, on the inner surface of which a certain part of the active substance deposits. The amount of the active substance in each of the 10 doses collected from one container, as well as in each of the 10 doses collected from 10 containers should be determined. The average value of the delivered dose and the uniformity of delivered dose should be calculated; the results should meet requirements stated in the specification. According to pharmacopoeial requirements large variability is permitted in regard to uniformity of delivered dose: the preparation complies with the test if 9 out of 10 results are between 75 % and 125 % of the average value and all are in range 65-135 % [1,6].
Test concerning uniformity of delivered dose is mandatory, and when it is conducted, the content of the active substance in the test solutions is the lowest compared to reference solutions for other tests. Taking into account the risks in relation to the correctness of the analysis results in testing of uniformity of delivered dose, validated procedures for the quantitative determination of active substances during this test were set out for the first time by the British Pharmacopoeia 2020 in the specific monographs [3,4].
It is necessary to ensure that each dose received by the patient has a therapeutic effect. The therapeutic effect of pMDIs depends on fine particle dose. However, according to the general articles 2.9.18 of the European Pharmacopoeia and the State Pharmacopoeia of Ukraine in order to determine fine particle dose on the relevant apparatus it is permitted to use more than one dose (but not more than 10), and the determined value should be divided by the number of actuations [1,6]. Leading pharmacopoeias and State Pharmacopoeia of Ukraine do not provide validated procedures for quantitative determination of active substances for this test and do not set out requirement to determine the uniformity of fine particle dose (UFPD). In general, the lower limit or lower and upper limits for fine particle dose are laid down in the specifications for particular preparations. Results for the individual doses and their uniformity are not estimated. Criteria for assessment of uniformity of fine particle dose are not established.
But there are much more factors that can affect the fine particle dose than the factors that affect delivered dose. These factors include the type of dispersed system (solution or suspension), the size of the suspended particles of the active substances, the aggregation of these particles, the content of propellant, ethanol and water, the nozzle length and actuator orifice diameter, the plume geometry, etc. [8,10,11]. The UFPD for the different pMDIs available on the market has not yet been studied. However, it is obvious that at a limit of ≥ 35 % and a result obtained of 35 % (which was determined as the total value for 10 doses divided by 10) the individual fine particle doses are less than 35 %.
Another problem should be identified. The test results for uniformity of delivered dose, obtained by the quantitative determination of active substance, do not characterize the volume of the metering chamber and the performance of the valve. For this purpose the test "Average dose mass and uniformity of dose mass" is necessary, which is not provided in the general article "Preparations for inhalation" of the European Pharmacopoeia [1] and in the guidelines on the pharmaceutical quality of inhalation and nasal preparations [2].
Previously, it was proposed to study of UFPD as the performance characteristic of pMDIs during their pharmaceutical development [12].
However, the rationale for the UFPD testing was limited only by the results of the study of model pMDIs containing suspensions; the advisability of this test for pMDIs with solutions was not considered. The choice of acceptance criteria for UFPD at the different stages of the preparation life cycle (during pharmaceutical development and full-scale manufacture) is still a problem.
In view of the above, the aim of the work was to substantiate the advisability of testing in regard to UFPD based on the results of pMDIs studies concerning different quality attributes that characterize the uniformity of dosing.

Research planning (methodology)
To achieve this goal, the main objects of the study were to be preparations in the form of pMDIs available on the market. Three preparations containing salbutamol sulfate (SS) in the form of a suspension, differing in the composition of excipients and the volume of the valve metering chamber were included in the study. Two of these preparations are listed in the Orange Book as Proair HFA aerosol, metered and Ventolin HFA aerosol, metered [13,14]. Also the preparation which contained beclometasone dipropionate (BD) 250 μg/dose as a solution and a similar model preparation which was equipped with the metering valve and the actuator from another manufacturer were chosen as the objects of the study. For these preparations, the average dose mass and uniformity of dose mass, average delivered dose and uniformity of delivered dose, as well as average fine particle dose and UFPD had to be determined. The sequence of dose sampling for all tests should correspond to that provided for testing of uniformity of delivered dose within one container (intra-inhaler) [1,6]. To determine delivered dose and fine particle dose it was necessary to use the appropriate analytical procedures for the quantitative determination of each active substance by high performance liquid chromatography (HPLC). Therefore, it was necessary to validate the analytical procedures for quantitative determination of SS and BD in the appropriate ranges [6,15]. For different preparations, it was also necessary to compare the particle size distribution of SS, which for suspension pMDIs might also significantly affect the fine particle dose [11,16]. At this step of the study, apparatus A (glass impinger) was chosen to determine the fine particle dose for one inhaler actuation. Total fine particle dose of active substance deposits in the lower chamber of the apparatus A that is more suitable for determination UFPD than multistage impactors. In the use of apparatus C, D, or E the fine particles dose is apportioned at the several collection plates/cups [1,6], which could significantly complicate the experiments required for this research.
Thereafter, it was necessary to perform the comparative assessment of the uniformity of dose mass, uniformity of delivered dose and UFPD and to compare these attributes regarding various preparations. According to the research results, the advisability of UFPD test during pharmaceutical development of pMDIs and the possibility of their standardization with regard to this attribute had to be substantiated. Weighing of samples was performed using analytical balances AUW 120D (Shimadzu, Japan). The solutions were prepared by mass-volume technique using volumetric amber glass flasks (class A, Simax, Czech Republic).

Materials and methods
Dose mass was determined according to the procedure: the protective cap was removed from the actuator, the container was shaken for about 30 seconds and one dose was discharged, after not less than 5 seconds container was shaken again and one dose was discharged. The container was removed from the actuator, valve stem was dried by filter paper (inside and outside). The container was weighed with the accuracy 0.1 mg (m 1 ). The container and the actuator were reassembled and shaken for about 5 seconds and one dose was discharged again. The container was removed from the actuator, the valve stem was dried by filter paper and the container was weighed again (m 2 ). The difference between m 1 and m 2 is the dose mass. The masses of the 1st, 2nd, 3rd, 99th, 100th, 101st, 102nd, 198th, 199th and 200th doses were determined sequentially. The average dose mass and the deviation (Δ, %) of the mass of each dose from the average dose mass were calculated.
Uniformity of delivered dose was determined according to the procedure described in the general article "Preparations for inhalation" of the European Pharmacopoeia [1] using the dose collection apparatus ERWEKA DUSA-MDI and the vacuum pump ERWEKA HBP 1000 with the flow rate meter DMF 2 (Erweka). The filter MN GF-4 Ø 25 mm (art. 414 0025, Macherey-Nagel GmbH & Co. KG) was used for dose collecting, which was placed on a filter-support base with an open-mesh filter support.
Fine particle dose was determined according to the to the procedure described in the general article "Preparations for inhalation" of the European Pharmacopoeia [1] using apparatus A (glass impinger). The mass balance was determined to ensure that test results were valid; the total mass of salbutamol in the both chambers should be not less than 75 % and not more than 125 % of the average delivered dose determined during testing for uniformity of delivered dose.
Water, carbon dioxide-free R was used as a solvent when preparations with SS were tested and methanol R was used in the case of preparations with BD.
HPLC studies were performed using Shimadzu Prominence-i LC-2030C 3D liquid chromatograph with a diode array detector (software: LabSolutions Lite version 5.82). Shimadzu Prominence-i LC-2030C 3D Plus liquid chromatograph with diode-array detector (software: Lab-Solutions version 5.93) was also used in the study of intermediate precision.
The content of salbutamol in the solutions was determined according to the analytical procedure described in the section "Related substances" of the monograph "Salbutamol Pressurized Inhalation" of the British Pharmacopoeia 2016 [4]. This procedure was validated in regard to quantitative determination of salbutamol in the appropriate range for such tests as uniformity of delivered dose and UFPD.
Analytical procedure. allow the chromatography to proceed for about 12 min.

Test solutions (TS
System suitability. The test is valid provided that: chromatographic column performance (apparent efficiency) calculated by the peak due to salbutamol in the chromatogram obtained with the reference solutions, is at least 4000 theoretical plates; for the peak due to salbutamol in the chromatogram obtained with the reference solutions the symmetry factor is 0.8 to 1.5; the maximum permitted relative standard deviation for the areas of peaks due to salbutamol in the chromatogram obtained withe the reference solutions does not exceed 2.11 % for three injection.
The potentiometric determination of pH (2.2.3) [9,12] of the buffer solution and the mobile phase were conducted using a pH meter Metrohm 827 lab (Metrohm, Switzerland) with an electrode Porotrode (Metrohm, Switzerland; cat. No. 6.0235.200).
The quantitative determination of BD in the solutions was performed according to the analytical procedure which was validated in the appropriate range for such tests as uniformity of delivered dose and UFPD.
Test solutions (TS). TS 1. Filtered solution of BD collected in the dose collection apparatus per discharge, in methanol R (~ 10 μg/ml).
TS 2. The solution of BD collected in the lower chamber of the apparatus A per discharge, in 50 ml of methanol R (about 2.5 μg/ml). allow the chromatography to proceed at least 15 min.

Reference solutions (RS
System suitability. The test is valid provided that: chromatographic column performance (apparent efficiency) calculated by the peak due to BD in the chromatogram obtained with the reference solutions, is at least 5000 theoretical plates; for the peak due to BD in the chromatogram obtained with the reference solutions the symmetry factor is 0.8 to 1.5; the maximum permitted relative standard deviation for the areas of peaks due to BD in the chromatogram obtained with the reference solutions does not exceed 2.11 % for three injection.
Validation of procedures for quantitative determination was carried out according to the generally accepted methodology [6,15]. Acceptance criteria for validation characteristics were calculated in accordance with the requirements of general article 5.3.N.2 of State Pharmacopoeia of Ukraine [6].
Determination of the particle size of SS was performed by laser diffraction [1,6] using the laser particle diffraction analyzer "Shimadzu SALD-2201" (Shimadzu; software: WingSALD-II, version 2.1.0). 20 ml of anhydrous ethanol was placed into the beaker, the container with preparation was shaken and the dose was discharged below the surface of ethanol; these actions were repeated 4 more times and the particle size in obtained suspension was immediately determined.

Research results
The performance characteristics of preparations for inhalation which are suspensions depend on the particle size distribution [8,10,19]. The data on the size particles distribution for SS in the pMDIs No. 1-3 are presented in Fig. 1 and Table 1.  Table 1 The maximum size of SS particle (D) in different fractions containing from 10 % to 90 % of particles from their total number in samples of suspensions from the pMDIs No. As can be seen from the data presented in Fig. 1 and in Table 1, the particle size distribution for SS was almost the same in the case of all three pMDIs.
The size of 99 % particles of SS should be not more than 10 μm and 90-95 % − not more than 5 μm. The content of particles with size up to 4.936 μm and up to 10.231 μm was (respectively): in the pMDI No. 1 -93,350 % and 99,684 %, in the pMDI No. 2 -90,016 % and 99,306 %, in the pMDI No. 3 -92,623 % and 99,688 %. Therefore, the particle size in the case of all pMDIs under the study met the well-accepted requirements.
The concentration range of the model solutions of SS for validation of the analytical procedure for quantitative determination was from 0.2 μg/ml to 2.8 μg/ml (equivalent to salbutamol). The concentration 0.7 μg/ml corresponds to the concentration in the test solution if the part (35 %) from the nominal dose of salbutamol (100 μg) deposits in the lower chamber of the apparatus A; the reference solution with the content of salbutamol 2.0 μg/ml was used when determination of delivered dose was performed. Fig. 2 shows the representative chromatograms obtained with placebo solution, reference solution 2 and test solution. Fig. 3 illustrates the chromatogram obtained with the model solution of salbutamol 0.23 μg/ml, which corresponds to the concentration in the test solution if the part (11.5 %) from the nominal dose of salbutamol (100 μg) deposits in the lower chamber of the apparatus A. The results in regard to the linearity, repeatability, accuracy and intermediate precision as well as acceptance criteria are summarized in Table 2. The data concerning stability of model solutions of SS are presented in Table 3.
The specificity of the analytical procedure was proved because the retention times (Rt) of the peaks corresponding to salbutamol in chromatograms obtained with the reference solution and the test solution had no difference, and the chromatogram with placebo solution had no peaks with the similar retention time (Fig. 2). The purity of the peaks due to salbutamol in the chromato-grams obtained with test solution and the reference solution, which was 1.000000, also confirmed the specificity of the analytical procedure. The signal-to-noise ratio (S/N) for the peak due to salbutamol in the chromatogram obtained with the model solution 0.23 μg/ml was 59.06 while the acceptance criterion for S/N is ≥ 10 (Fig. 3).
According to the results of validation studies, the analytical procedure for the quantitative determination of salbutamol by HPLC in the appropriate range met the requirements for the linearity, repeatability, accuracy and intermediate precision against the acceptance criteria calculated for tolerance B=10 % ( Table 2).
The results of validation studies with using two chromatographic columns proved that the test model solutions of SS met the requirements for their stability (Table 3).   Validation of the analytical procedure for the quantitative determination of BD was performed using model solutions in concentration range from 0.5 μg/ml to 14.0 μg/ml. The concentration of BD of 1.25 μg/ml cor-responds to its content in the test solution if the part (25 %) from the nominal dose 250 μg deposits in the lower chamber of the apparatus A; the reference solution with the content of BD 10.0 μg/ml was used when determination of delivered dose was performed. Fig. 4 shows the representative chromatograms obtained with placebo solution, reference solution 2 and test solution. Fig. 5 illustrates the chromatogram obtained with the model solution of BD 0.5 μg/ml, which corresponds to the concentration in the test solution if the part (10 %) from the nominal dose of BD (250 μg) deposits in the lower chamber of the apparatus A. The results in regard to the linearity, repeatability, accuracy and intermediate precision as well as acceptance criteria are summarized in Table 4. The data concerning stability of model solutions of BD are presented in Table 5. The specificity of the analytical procedure was demonstrated because the retention times (Rt) of the peaks corresponding to BD in chromatograms obtained with the reference solution and the test solution did not differ, and in the chromatogram obtained with placebo solution there was no peak with the similar retention time (Fig. 4). The purity of the peaks corresponding to BD in the chromatograms obtained with test solution and the reference solution, which was 1.000000, also confirmed the specificity of the analytical procedure. The signal-tonoise ratio (S/N) for the peak corresponding to BD in the chromatogram obtained with the model solution 0.5 μg/ml was 30.81 while the acceptance criterion for S/N is ≥10 (Fig. 5).  According to the results of validation studies, the analytical procedure for the quantitative determination of BD by HPLC in the appropriate range met the requirements for the linearity, repeatability, accuracy and intermediate precision against the acceptance criteria calculated for tolerance B=5 % (Table 4).
The results of validation studies using two chromatographic columns proved that the test model solutions of BD met the requirements for their stability (Table 5).
In the experiments using both analytical procedures the chromatographic systems met the requirements to their suitability and minor changes of the chromatographic conditions did not affect the parameters of the suitability; thus the robustness of both procedures was proved (Tables 6, 7).
The results of the tests in regard to dose mass and uniformity of dose mass for the pMDIs with SS are presented in Table 8. Table 9 shows the results of the same tests for the pMDIs with BD.

Note. ∆deviation from the average mass
The results of the tests in regard to delivered dose and uniformity of delivered dose for the pMDIs with SS are given in Table 10. Table 11 shows the results of the same tests for the pMDIs with BD.
The results of the tests in regard to fine particle dose and uniformity of fine particle dose for the pMDIs with SS are given in Table 12. Table 13 shows the results of the same tests for the pMDIs with BD.   Table 12 The results of the tests in regard to fine particle dose (FPD) and uniformity of fine particle dose (UFPD) for the pMDIs with salbutamol sulfate (SS) No.  Table 13 The results of the tests in regard to fine particle dose (FPD) and uniformity of fine particle dose (UFPD) for the pMDIs with beclometasone dipropionate (BD) . For study of these characteristics, analytical procedures for quantitative determination of SS (equivalent to salbutamol) and BD were used, the correctness of which had been proved by the results of validation studies (Fig. 2-5, Tables 2-7).
The test pMDIs were different in their average dose mass of (Tables 8 and 9) because of the different metering chamber volumes of the valves used for the preparations, as well as the different density of the content of the container, which depended on the formulations of these pMDIs. For the single container, the deviations of the mass of individual doses from the average value were minor and did not exceed 1.5 %, and the relative standard deviation (RSD) did not exceed 1.0 % (Tables 8 and 9). However, there is a risk of variability in the volume of the metering chamber in different batches of valves; it should be taken into account when limits for average dose mass and uniformity of dose mass are established. For example, in the case of the pMDI No. 1 the average dose mass for the batch AET46A was 30.59 mg, for the batch AFB40A it was 32.45 mg and for the batch AFC39A it was 33.15 mg. Therefore, the difference in average dose mass between the first batch and the next two batches was 6.1 % and 8.4 %, respectively. Thus, the limits for the average dose mass and uniformity of dose mass should be established in the specifications for pMDIs, although these tests are not provided as obligatory by the leading pharmacopoeias, State Pharmacopoeia of Ukraine and EMEA guidelines [1-3, 5, 6]. These limits should be established during pharmaceutical development using the appropriate valves of different batches.
In contrast to the average dose mass, delivered dose should be standardized by the content of active substance. The average values of delivered dose in the case of the pMDI with SS were close (Table 10) although the differences in regard to the average dose mass were observed (Table 8). For the single container, the variability of delivered dose was much more significant than the variability of average dose mass, that was evidenced by RSD value, which was the almost 10 times higher, as well as by the deviations of individual delivered doses from the average delivered dose, which were up to 10-15 %. The highest value of RSD for delivered dose was found in the case of the pMDI No. 2 without ethanol in the formulation, and the lowestfor the pMDIs with BD, which were solutions. At the same time, the higher content of ethanol in the pMDIs No. 5 and No. 6 probably led to the decrease in the average delivered dose of BD compared to the pMDIs with SS (Tables 10 and 11) [19,20].
If the dose mass mainly depends on the volume of the valve metering chamber, then the delivered dose depends on the metered dose and the design of the actuator as well, where part of active substance might deposit.
In view of the above, it is clear that even in the case of pMDI-solutions, the test in regard to the uniformity of dose mass cannot replace the test concerning uniformity of delivered dose and vice versa, and limits for the uniformity of dose mass and limits for uniformity of delivered dose cannot be the same (9 out of 10 results should lie between 75 % and 125 % and all should lie between 65 % and 135 %). Unfortunately, according the provisions that are laid down in the general articles "Dosage forms for inhalations" and "Aerosols and sprays" of the State Pharmacopoeia of the Russian Federation (SP RF) it is acceptable to replace the test "Uniformity of delivered dose" by the test "Uniformity of dose mass" in the case of the pMDI which are solutions [21]. For pMDIsolutions it is not acceptable to assess the uniformity of delivered dose by the value of average dose mass and the differences between average and individual masses because these two tests are intended to determine the different attributes that are content of active substance and mass; moreover, these attributes depends on different factors and reasons for non-conformity could be different. Furthermore, according to the general article 2.9.18 of the European Pharmacopoeia [1], during testing for the aerodynamic assessment of fine particle the average value of delivered dose should be used as the acceptance criterion to calculate mass balance, which serves to ensure that test results are valid. However, the mass balance is not stipulated in the general article "Aerodynamic distribution of fine particles" of the SP RF in the case of using the apparatus A, C, D and E for testing of pMDIs [21].
The delivered dose characterizes the suitability of the actuator and does not ensure the therapeutic effect which depends on the fine particle dose. The same nominal doses and almost the same delivered doses do not indicate an equivalent therapeutic effect of the two pMDIs if they differ in fine particle dose [22]. However, the limits in regard to fine particle dose for the pMDI with the various active substances as well as the acceptance criteria for uniformity of fine particle dose are not established by leading pharmacopoeias, State Pharmacopoeia of Ukraine and SP RF [1,3,5,6,21].
The lower limits for the fine particle dose, which are of ≥ 35 % and ≥ 25 % of the nominal dose, are established in the specifications for the pMDIs with SS and BD respectively. For the three tested pMDIs with SS it was found that despite the same particle size distribution of SS and almost the same average delivered dose the fine particle doses were significantly higher in the case of the pMDIs No. 1 and No. 3 compared to the pMDI No. 2. This was due to the low ethanol content in the pMDIs No. 1 and No. 3 [19,20].
It is necessary to ensure that each dose could provide an appropriate therapeutic effect, but delivered dose is not the comprehensive characteristic that could guarantee the therapeutic efficacy of the pMDI. Therefore, uniformity of fine particle dose should be determined and standardized. The data presented in Tables 12 and 13 shows that all tested fine particle doses met the requirements from the first dose to the last one stated on the label. The level of the uniformity of fine particle dose and the level of the uniformity of delivered dose were approximately the same, as it demonstrated by the very close values of RSD. The highest deviations of individual fine particle doses from the average value did not exceed 10-15 % in the case of the pMDIs with SS and 7-10 % in the case of the pMDIs with BD. The dosing of the pMDIs with solutions of BD was more uniform considering the RSD values.
During pharmaceutical development [2,23] it should be demonstrated that an average fine particle dose exceeds the established lower limit at least on the value of the highest deviation of individual fine particle doses from the average dose . The testing for uniformity of fine particle dose could be a tool for long-term study of the suspension stability in the containers because the aggregation of particles results the decrease of the fine particle dose [8,16,19]. The constant level of the uniformity of fine particle dose within the shelf life is a guarantee of the effectiveness of each dose of the pMDIs during their storage [8,12].
The values of the 10 results for testing of fine particle dose should be higher than the lower limit for this characteristic. All 5 tested pMDIs met this requirement (Tables 12 and 13). If lower and upper limits for fine particle dose are established in the specification (for example, from 18 % to 33 %), the all 10 results of testing for fine particle dose should be in this range. In the case of the pMDIs with suspensions of SS, the content of the active substance in each of the 10 doses of fine particle was within ±15 % of the average value, and in the case of the pMDIs with solutions of BD the content of BD was within ±10 % of the average value (Tables 12 and 13). These data could be used for the standardization of the uniformity of fine particle dose.
The results of research create a basis for the determination of uniformity of fine particle dose using multistage impactors [1,6,9]. It is also rational to conduct test for UFPD in regard to pMDIs if the lower and upper limits for fine particle dose are established in the specifications, as well as using 10 different containers of the same pMDI (inter-inhaler testing).
Highly sensitive analytical methods should be used to determine UFPD. Therefore, it is rational to develop or improve procedures for quantitative determination of active substances, in particular, SS or formoterol fumarate by HPLC using a fluorescence detector, etc. [24,25].
The obtained results could be used in the development, research and standardization of preparations in the form of pMDI, as well as they could be considered for development of the general articles and monographs of State Pharmacopoeia of Ukraine.

Conclusions
The validation of two analytical procedures for the quantitative determination of SS and BD by HPLC in the suitable concentration ranges including low content of these substances was conducted. The 3 pMDIs with SS and the 2 pMDIs with BD were studied. It was shown that for the three pMDIs with SS the particle size distribution in the containers and the average delivered dose were almost the same, but these preparations were different in the average dose mass and fine particle dose. According to the research results, the expediency of the determining of average dose mass and testing for the uniformity of dosing by the dose mass and fine particle dose was substantiated. It was demonstrated that dosing was more uniform in the case of the pMDIs with solutions compared to the pMDIs with suspensions. Approaches of leading and other pharmacopoeias to the uniformity of dosing of pMDIs have been critically discussed. The expediency of the testing for UFPD during pharmaceutical development was substantiated because it is the fine particle dose that determines the therapeutic effect of pMDIs. The issues concerning the standardization of UFPD for pMDIs were discussed.

Conflict of interests
The authors declare that they have no conflicts of interest.

Financing
The study was performed without financial support.