Samples were collected from the capital, Phnom Penh (urban area), and Svay Rieng and Kandal provinces (rural areas). The list of licensed outlets in Phnom Penh was obtained from the DDF, MoH of Cambodia, in June 2013. The calculated sample size was 193 pharmacies, 58 depot A outlets, and 79 depot B outlets, with an alpha value of 5 and power of 0.8. In Phnom Penh, as many private drug outlets as possible of the four types of private drug outlets (i.e., pharmacies, depot A, depot B, and non-licensed outlets) were visited within the period of sample collection. For sampling purposes, a stratified random scheme developed by means of random number tables was used. All the outlets found in Svay Rieng and all illegal outlets found in the sampling area were visited. Additionally, some samples were collected from wholesalers and drug outlets in Kandal province while the sampling teams moved between Phnom Penh and Svay Rieng. In Cambodia, a pharmacy outlet is run by a registered pharmacist, a depot A outlet by an assistant pharmacist (with 4 years’ pharmacy training), and a depot B outlet by a retired midwife or nurse . The sampling areas were selected in consultation with the MoH, taking into account the degree of urbanization, population density, concentration of drug outlets, budgetary limitations, and geographic importance in sharing a border with another country.
Cefixime tablets, omeprazole capsules, co-trimoxazole (a combination of sulfamethoxazole and trimethoprim) tablets, clarithromycin tablets, and sildenafil tablets were selected as candidate medicines in consultation with the DDF. Cefixime, omeprazole, clarithromycin, and sildenafil were chosen in 2010 as newer-generation medicines; this was in contrast to selection from a list of essential medicines in our previous studies [15, 16]. Sample information was collected using a sampling form that included the contents of the packages sold, price, and outlet information. Samples were collected between June 7 and 15, 2010 by two teams. Each team consisted of a research investigator, a locally recruited sampling officer, and a sampling assistant. The locally recruited members were provided with training before sampling and instructed to purchase medicines. The sampling officer purchased medicines in an outlet and completed a sampling form for each sample. Medicines collected from the same outlet and labeled with the same international non-proprietary name, brand name, strength, size, batch/lot number, and manufacturing and expiry dates were considered one sample. For authentication purposes, the teams collected containers or packages for most of the samples. Samples were preserved at 20–25°C until analysis.
Details of the packaging condition and the label information of the samples were carefully noted. Compliance with the Association of Southeast Asian Nations Common Technical Dossier (ACTD) for the registration of pharmaceuticals for human use (to which drug registration in Cambodia conforms) was examined . Bar codes were also recorded.
Materials for quality evaluation
United States Pharmacopeia (USP) reference standards of omeprazole, sulfamethoxazole, trimethoprim, and clarithromycin-related compound A (6,11-di-o-methyl erythromycin A) were purchased from the Reference Standard Center, Bureau of Drugs and Narcotics, Thailand (Nonthaburi, Thailand). Reference standards of cefixime, clarithromycin, and sildenafil citrate were generously donated by Astellas Pharma Inc. (Tokyo, Japan), Taisho Toyama Pharmaceutical Co, Ltd. (Tokyo, Japan), and Pfizer Japan Inc. (Tokyo, Japan), respectively. Metronidazole and butyl p-hydroxy benzoate were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Primidone and sulfadoxine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Lansoprazole was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Methanol and acetonitrile of high-performance liquid chromatography (HPLC) grade was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals were commercially available and of analytical grade.
The Medicine Quality Assessment Reporting Guidelines (MEDQUARG) were followed when reporting in generally .
To assess the pharmaceutical quality of the samples, active ingredients of the samples were quantified by HPLC using ultraviolet detection (Shimadzu, Kyoto, Japan). The system suitability for analysis of each medicine was verified according to USP 30. A linear relationship between the peak area and concentration of each reference standard was observed within the range of 25–200% of the active ingredient (r2 = 0.999–1.000), and the assay was performed within that range. The intra- and inter-day coefficient of variation was less than 3.0%. In addition, the methods were validated as being repeatable and accurate (n = 6). Metronidazole, butyl p-hydroxybenzoate, primidone, lansoprazole, and sulfadoxine were used as internal standards in the analysis of cefixime, clarithromycin, co-trimoxazole, omeprazole, and sildenafil, respectively. The samples were analyzed between July 2010 and March 2012 in Kanazawa University. The quality evaluation was completed within the expiry date for each sample.
For cefixime tablets, omeprazole capsules, co-trimoxazole tablets, and clarithromycin tablets, the assay, a content uniformity test, and dissolution test were performed with reference to USP 30, USP34, or British Pharmacopeia (BP) 2010 as indicated on the package insert or outer package.
For sildenafil tablets, the assay was performed according to the method described previously . In each sample, three or six tablets were analyzed. The acceptable range was set as follows: in the quantity test, sildenafil tablets containing not less than 90% and not more than 110% on an average quantity taken from 10 units, and no unit less than 75% or more than 125% of the labeled amount of sildenafil; in the dissolution test, the acceptable range was an average dissolution rate in 3 or 6 units equal to or greater than 75% and no unit less than 50%, with a dissolution time of 15 minutes. The content uniformity test could not be conducted because of insufficient material.
The methodology of the authenticity investigation and registration verification was adopted from the World Health Organization [15, 16, 20, 21]. Label information on the packages and containers was cross-checked with a database prepared after collection. Photographs of each sample, its packaging, and package inserts were obtained for such purposes. These data were then catalogued. A database of manufacturer addresses was also prepared using printed information, Web searches, and e-mail and telephone communication. Portions of all samples were then sent to the respective manufacturers, requesting verification of their products. Information on the manufacturers and their medicines was requested from medicine regulatory authorities of the countries in which the manufacturing took place. Furthermore, each sample’s registration was confirmed by the DDF.
Considering the limitations of the small sample size, descriptive analysis was performed using SPSS 19.0.0 (IBM SPSS Inc, Chicago, IL, USA). Where appropriate, Fisher’s exact test was used to test the significance of categorical variables. Statistical significance was evaluated at the 5% level.