Institutional ethical approval was not needed for this study as it is does not involve human subjects, although good ethical practice for such studies has been suggested by Tabernero et al., to maintain the privacy and confidentiality of the surveyors and the surveyed . Regulatory approval was given by the respective countries’ Medicine Regulatory Authorities (MRAs), and annual reports have been submitted to them.
Study design and sample collection
As suggested by the regulatory authorities of the respective countries, pioglitazone was chosen as a target medicine because of the past history of similar medicines to show problems in the dissolution test. The medicine also appears in the essential drug list of Myanmar. The design and analytical methods used in this study followed as far as possible the guidelines of the WHO and those proposed by Newton et al. [27, 28]. Cross-sectional sampling with the mystery shopper approach was used. In each sampling location, the initial sampling plan was to follow a random sampling protocol, though in practice this was not always possible due to the unavailability of medicine, availability of insufficient quantities, incomplete list of shops, or closure of a listed shop at sampling sites [28, 29]. Samples were collected with prescriptions from hospitals and clinics of Huangpu District and Pudong New Area of Shanghai, China, between October and December in 2012. Personally imported samples purchased via internet sites were collected based on the availability of commercial brands in the site during September and December, 2013. Google Japan was used as a search engine to find sites; the search term was ‘ピオグリタゾン AND 個人輸入’ for Japanese language sites and ‘Pioglitazone and personal import’ for English language sites. In Myanmar, samples were collected without prescription from shops in Yangon during October 2015. The samples were purchased by the mystery shoppers without any preference to specific brands in an attempt to purchase as wide a variety of available commercial brands as possible. Medicines collected from the same shop/site and labeled with the same international non-proprietary name (INN), strength, size, brand name, batch/lot number, and manufacturing and expiry dates were considered as one sample. The maximum number of samples collected from each shop was three.
Chemical assessment of the quality of pioglitazone tablets purchased in all the sampling location was carried out at the laboratory of Kanazawa University, Japan. Every sample was placed in an individual ziplock bag together with the recoded data, and securely stored in an air-conditioned laboratory (20–25 °C) until analysis. Analysis of all samples was carried out before the stated expiry date. The analysis consisted of observation, authenticity and legality investigation, registration verification, pharmacopoeial analysis (identification, potency, content uniformity and dissolution test) and dissolution profiling.
Each sample was given a unique code after the shipment was received. Details of the packaging condition and label information were noted carefully. Observations included the packaging and labeling, and physical appearance of the tablet (size, shape, color, etc.) according to the WHO guideline and the International Pharmaceutical Federation (FIP) checklist for visual inspection of medicines [28,29,30]. For personal import samples shipped to Japan from internet pharmacies, the websites were checked for compliance with the Pharmaceutical Affairs Law of Japan [31, 32].
Authenticity investigation and legitimacy verification
For the authenticity investigation of the products and legitimacy verification of the manufacturers, a detailed questionnaire was sent to each manufacturer and regulatory authority of the manufacturing country. Sample questionnaire for product authentication and legitimacy verification are presented in Supplemental File S1 and Supplemental File S2. Each questionnaire contained detailed information about the product, including manufacturer, batch number, manufacturing and expiry dates, and dosage and strength of the product, as indicated by WHO and other related studies [17, 28, 29, 33]. The registration status of all products as stated on the product packaging was recorded, and included on a questionnaire sent to the importing country to confirm the registration of the product and manufacturer (sample registration verification form is presented as Supplemental File S3).
Pioglitazone hydrochloride as a reference standard, benzophenone as an internal standard, methanol, acetonitrile, ammonium acetate, potassium chloride and other chemicals of reagent grade were procured from the Wako Pure Chemical Industries Ltd. Japan. Hydrochloric acid was purchased from Nacalai Tesque Inc. and acetic acid from Alfa Aesar. Analysis of the sample was done by high-performance liquid chromatography (HPLC) according to the modified and validated JP (Japanese Pharmacopoeia) protocol [34, 35], using a Shimadzu Prominence HPLC equipped with a Phenomenex Gemini NX C18 column (150 × 4.6 mm) and a UV-photodiode array detector (SPD-20A/20AV Series). The flow rate, injection volume, and detection wavelength were kept unchanged throughout the entire analysis. The dissolution test was performed using 900 mL of a solution for each of the units with an NTR-VS 6P dissolution apparatus (Toyama Sangyo Co. Ltd., Osaka, Japan). The dissolution medium was prepared by mixing 50 mL of 0.2 mol/L hydrochloric acid and 150 mL of potassium chloride solution, adding water to make 1000 mL, and adjusting to pH 2.0 with 5 mol/L hydrochloric acid. Drug release studies were carried out according to the United States Pharmacopoeia (USP) Type II dissolution apparatus paddle method. The paddle was set to rotate at 50 rpm (revolutions per minute) for 45 min and the temperature was maintained at 37 ± 0.50 °C. Standard solutions were prepared by dissolving accurately weighed quantities of pioglitazone hydrochloride (reference standard) and benzophenone (internal standard) in the diluent to obtain concentrations of 0.2 mg/mL and 0.1 mg/mL, respectively. Serial dilutions were made to 0.025 mg/ml. The concentration of the test solution was kept at 0.1 mg/ml. The relationship between the peak area and concentration of each reference standard was linear within the range of 25–200% of the active ingredient (r2 = 0.999–1.000), and the quality test was performed within that range.
Samples were evaluated as meeting the quality specifications if the amount of active pharmaceutical ingredient (API-pioglitazone hydrochloride) in each of the units, as determined by the content uniformity test, lay within the range of 95.0–105.0% of the label claim. For content uniformity, the acceptance value (AV≦15.0) was calculated according to USP 34 . In the dissolution test, Q = 80% or more was used as the criterion of acceptability as indicated by the pharmacopoeia [34,35,36].
Descriptive statistical analysis was performed using Microsoft Excel.