Introduction
Counterfeit drugs are a global problem with significant and well documented consequences for global health and patient safety, including drug resistance and deaths. This multibillion-dollar industry exists beyond geopolitical borders, and threatens public health in both developed and developing countries and regions.1-3 Cefuroxime axetil, an antibiotic, and Ethambutol hydrochloride (HCI), an antituberculosis medicine, are two essential medicines that are commonly counterfeited, leading to treatment failure and often death.3, 5
The substandard medicines, herbals, and counterfeit medicines in the Vietnamese market in the year of 2011 are 2.81%, 6.09%, and 0.1%, respectively, determined by the Vietnam National Institute of Drug Quality Control (NIDQC). In the NIDQC laboratories, routine analytical methods used to detect suspicious substandard and counterfeit medicines include: HPLC, LC-MS, TLC, HP-TLC, UV-Vis, GC, GC-MS, AAS, and others. The sample preparation step is critical for all these methods, but setup of this step is very time-consuming and has to be performed in a traditional wet laboratory setting.6
Modern spectroscopic technologies make complex analysis possible through detection of suspicious counterfeit or substandard products with minimum or no sample preparation, and without a traditional laboratory environment. With appropriate spectroscopic analyzers, multiple samples can be screened simultaneously and analyzed against reference spectra, if available. Chemometrics has been widely used in spectroscopic data analysis, where a wealth of information can be obtained from drug samples with no prior knowledge. However, this mode of detection requires expertise to build and manage chemometric models.4, 7-12
The portable and handheld vibrational spectroscopic technologies, including Raman, Near Infrared (NIR), and Fourier Transform Infrared (FTIR), have advanced tremendously in recent years. Together with spectral libraries and other portable technologies, such as portable X-ray Fluorescence (XRF) and GC-MS, these analyzers realize the transition from post-screening in a traditional laboratory to near or real-time screening in a field-based laboratory.
This study explored an orthogonal approach of using multiple analytical technologies of handheld Raman, handheld NIR and portable FTIR analyzers to rapidly detect the counterfeit drug products of Cefuroxime axetil and Ethambutol HCl from Vietnam.
Experimental
Materials
Ethambutol HCI reference standard (RS) (Lot Number: 0105188) and Cefuroxime axetil RS (Lot Number: 021067.02) were provided by the Vietnamese Pharmacopeia Commission. Tablets and capsules for both drug products (Tables 1-4) were prepared by the Research and Development Division of Vietnam NIDQC for their internal testing only. The corresponding placebo tablets and capsules were prepared based on the formula of both products legally registered in Vietnam. All tablet samples were grinded before use.
Table 1. Ethambutol Tablets (400 mg) and Placebo Ethambutol Tablets
Table 2. Ethambutol Capsules (100 mg) and Placebo Ethambutol Capsules
Table 3. Cefuroxime Tablets (600 mg) and Placebo Cefuroxime Tablets
Table 4. Ethambutol Capsules (100 mg) and Placebo Ethambutol Capsules
Instruments
Both handheld Raman and handheld NIR analyzers were chosen for pharmaceutical raw material identification and finished products screening, while portable FTIR analyzer with Attenuated Total Reflectance (ATR) module provided quantitative and qualitative analysis for all samples.
Methods
Handheld Raman analyzer: Raman spectra were acquired on a handheld Raman analyzer using the excitation wavelength at 785 nm. The reference spectra of active pharmaceutical ingredients (API) (Ethambutol HCI and Cefuroxime axetil) and finished products (Grinded Ethambutol HCl tablets [400 mg]; CT1 260414) were collected from the corresponding reference standards (RSs) in the 4-mL glass vials. All samples (grinded tablets and capsule powders) were tested against the reference spectra of RSs and CT1 tablets.
Handheld NIR analyzer: The reference spectra of API (Ethambutol HCI and Cefuroxime axetil) and finished products (grinded Ethambutol tablets [400 mg]; CT1 260414) were collected from the corresponding reference standards in 4-mL glass vials. All samples (grinded tablets and capsule powders) were tested against the reference spectra of RSs and CT1 tablets.
Portable FTIR-ATR analyzer: The instrument was turned on for 1 hour until the “Green” status and the energy level around 26,000 counts displayed in the diagnostic window. The method was set up as “Data collect only” with the Spectrum Range: 4000-650 cm-1; Background Scans: 32; Sample Scans: 32; Apodization Method: HapoGenzel; Instrument Library: disabled. The ATR surface cleanliness was monitored by MicroLab PC software (Version v4.5.93.0). A drop of sample was placed on the top of ATR surface, then FTIR spectrum was collected in the absorbance mode with a single bounce ATR. Spectra were collected in the range of 4000-650 cm-1 (full range) and 1800- 1150 cm-1 (fingerprint region) for RSs, finished tablets CT1, and placebo tablets CT1 in Qualitative Search mode. A reference spectral library was established from all the reference spectra. All sample spectra were compared against the reference spectra in the library and a hitquality index (HQI) value (between 1.00 and 0) was used to indicate the correlation between the reference spectra and the sample spectra.
Results and Discussion
Handheld Raman Analyzer
The results from handheld Raman analyzer are shown in Tables 5-7. “Pass” results were given when a positive match was discovered by the instrument with a statistical assessment of P>0.05 (within the 95th percentile), while “Fail” results were given when no positive match was discovered by the instrument with a statistical assessment of P<0.05 (outside of the 95th percentile).
Table 5. Results of Ethambutol Tablets (400 mg) against Ethambutol RS and CT1 Methods
Table 6. Results of Placebo Ethambutol Tablets against Ethambutol RS Method
Table 7. Results of Cefuroximel Tablets (600 mg) against Cefuroximel RS Method
When both API RSs (Ethambutol HCl and Cefuroxime axetil) were tested against the RS reference spectra, “Pass” results were shown, but “Fail” results were given for all finished products. These rapid screening and analysis results indicate that when using the reference spectra of API RSs, this handheld Raman analyzer can report suspicious counterfeited or contaminated raw APIs, but is not suitable for the finished products, due to the interference of various excipients.
Only Ethambutol HCl tablets CT1 and CT3 gave “Pass” results when all tablets samples (CT1-CT5) were tested against the authentic finished product Ethambutol HCl tablet CT1 reference spectra. Based on Table 1, only CT3 contains Lactose, which contributes significantly in Raman spectra for CT1 and CT3, while CT2, CT4, and CT5, which contain no Lactose but do contain excipients similar to CT1, such as Wheat Starch or HPMC, failed in finding a positive match against CT1 method.
These preliminary results from Ethambutol tablets demonstrate a possibility of “False Negative” in this handheld Raman instrument, if using a finished product by one manufacturer to test against the same finished products, but by different manufacturers with different formulations and matrices of excipients. These complications can interfere extensively with the Raman signal at the excitation of 785 nm. Though more testing needs to be explored, it is recommended that establishing the reference spectra for finished products by the same manufacturer, as long as the formulation is consistent.
Handheld NIR Analyzer
The results from handheld NIR analyzer are listed in Tables 8-10, which provide similar results as the handheld Raman analyzer. Rapid screening by both handheld Raman and handheld NIR analyzers can be complementary and beneficial for the validation purpose.
Table 8. Results of Ethambutol Tablets (400 mg) against Ethambutol Tablet RS and CT1 Methods
Table 9. Results of Placebo Ethambutol Tablets against Ethambutol RS Method
Table 10. Results of Cefuroxime Tablets (600 mg) against Cefuroxime RS Method
Portable FTIR Analyzer
The results from portable FTIR analyzer using ATR module are listed in Tables 11 and 12. All spectra were acquired in the full wavelength region of 4000-650 cm-1. The reference spectra library was established from the reference spectra of Ethambutol HCl RS, Ethambutol tablet CT1, Cefuroxime axetil RS, and Cefuroxime tablet CT1. While all authentic tablets and capsules for both medicines (CT1-CT5) from different manufacturers returned with HQI values >0.95, demonstrating a statistical assessment within the 95th percentile, all Placebo tablets and capsules for both medicines returned with HQI values <0.95 but still >0.50. This demonstrates possible rapid detection of samples that contain no APIs using portable FTIR-ATR instrument.
Table 11. HQI of Ethambutol Tablets, Capsules, and Placebo Tablets, Capsules
Table 12. HQI of Cefuroxime Tablets (400 mg) and Placebo Tablets
The fingerprint region of 1800-1150 cm-1 was selected for identifying APIs of Ethambutol HCl and Cefuroxime axetil in the finished products, due to specific absorption detect suspicious counterfeit finished products more effectively than handheld Raman and NIR analyzers.
Table 13. HQI of Ethambutol Tablets, Capsules, and Placebo Tablets, Capsules (1800-1150 cm-1)
All Cefuroxime tablets analyzed returned with HQI values >0.98 against the reference spectral library established from Cefuroxime axetil RS, Tablet CT1, and Placebo Tablet CT1, while the placebo products returned with HQI values <0.01, listed in Tables 14-17. The portable FTIR-ATR instrument has shown robust results to identify Cefuroxime axetil API rapidly in all finished products using the fingerprint region of 1800-1150 cm-1.
Table 14. HQI of Cefuroxime Tablets (600 mg, 1800-1150 cm-1)
Table 15. HQI of Placebo Cefuroxime Tablets (1800-1150 cm-1)
Table 16. HQI of Cefuroxime Capsules (120 mg, 1800-1150 cm-1)
Table 17. HQI of Placebo Cefuroxime Tablets
Summary
An orthogonal approach using three different vibrational spectroscopic technologies—handheld Raman, handheld NIR, and portable FTIR instruments—for rapid detection of counterfeit drug products was explored in this study. Both handheld Raman and NIR analyzers report “False Negative” results using one manufacturer’s finished product testing against different manufacturers’ finished products. This suggests the strong interference of the “inactive ingredients”: excipients in the finished products actually can play a very active role in the rapid screening by Raman and NIR. The portable FTIR instrument using ATR module provides an effective way to rapidly screen counterfeit finished products containing no active pharmaceutical ingredients.
Acknowledgements
The authors thank James R. Austgen, PhD, Writer-Scientific at USP, for editorial contributions.
Author Biographies
Bei Ma provides scientific and business development expertise in identifying and pursuing business opportunities that support the public health mission and global strategy at the United States Pharmacopeial Convention (USP). Her focus has also been in developing and implementing USP global public health initiatives and programs in East Asia and Asia Pacific Region. Prior to joining USP, Bei worked as Senior Research Scientist and Global Market Specialist at Mettler-Toledo International. She currently serves at the organizing committee for IFPAC® – ASIA 2014 and recently served as a scientific board member and co-chair for Food Safety Session at the 5th Annual IFPAC® - 2014 Food Quality, Safety & Analysis Symposium. She is a member of American Chemical Society and Society of Applied Spectroscopy.
Le Thi Thien Huong has been working at National Institute of Drug Quality Control of Vietnam (NIDQC) since 1998. She is a senior researcher in the Physicochemical Lab and Medicinal Material Lab and Member of Expert Group of Vietnamese Pharmacopoeia Commission. She has a Bachelor’s degree and a Master’s degree in Pharmacy from the Hanoi University of Pharmacy. She is the author of 10 articles published in Vietnam Journal of Drug Quality Control and co-author of 4 projects of Vietnam Health Ministry for Development of the HPLC analytical methods. She attended USP Visiting Scientist Program, and worked with USP Spectral Library Group from May 1, 2014 to May 30, 2014.
Dr. Yong Liu is a research scientist at USP. He received a PhD in Analytical Chemistry from Peking Union Medical College (PUMC) in China. Before joining the USP, Dr. Liu was a Postdoctoral fellow at the University of Maryland, Baltimore. In his current position, he utilizes both handheld and bench top spectroscopic methods to study the quality of pharmaceutical, food, and dietary supplement products.
Magdy M. Kamel has been participating to the Visiting Scientist Program (VSP) at USP for nine months since October 2013. He is a manager of the methodology department at European Egyptian Pharmaceutical Industries. He has over 18 years of experience in QC, stability study programs, and method developing and validation. He is working on his master's in quantum dots nanocrystal, Alexandria University.
Ed Zhao is Vice President, Global Education & Training for USP. He is responsible for pharmacopeial-based education programs worldwide. Ed has had progressively responsible positions within the organization, including Vice President of International Business Development, Allied Compendial Programs in identifying new opportunities, forming partnerships and alliances with organizations, developing business plans, and executing strategies to develop USP’s business and science interests globally. He has also served Regional Champion in East Asia, working closely with ministries of health, pharmacopeias, and other stakeholders in China, Korea, Japan, and the Association of South East Asian Nations. Before joining USP in 2004, Ed held many positions in the pharmaceutical industry in the U.S., Canada, and China, including Marketing Director for Asia Pacific for Chattem Inc., Senior Account Director for McCann Erickson Healthcare Worldwide, and Marketing Manager for Johnson & Johnson companies in Ontario, Pennsylvania, and Beijing. Ed received his Bachelor of Engineering in chemical engineering from East China University of Chemical Technology and his MSc in pharmacology from the University of Saskatchewan. Ed has an MBA in international business and finance from McGill University.
References
- WHO. Combating counterfeit drugs: a concept paper for effective international collaboration. Draft. http://www.who.int/entity/medicines/services/counterfeit/ CombatingCounterfeitDrugs-Conceptpaper.pdf. Accessed July 16, 2006.
- Cockburn R, Newton PN, Agyarko EK, Akunyili D, White NJ. The global threat of counterfeit drugs: why industry and governments must communicate the dangers. PLoS Med 2005;2:e100.
- Wondemagegnehu E. Counterfeit and substandard drugs in Myanmar and Viet Nam. WHO Report. WHO/EDM/QSM/99.3. Geneva: WHO, 1999.
- Gryniewicz-Ruzicka CM, JRodriguez JD, Arzhantsev S, Buhse LF, Kauffman JF. Libraries, classifiers, and quantifiers: A comparison of chemometric methods for the analysis of Raman spectra of contaminated pharmaceutical materials. Journal of Pharmaceutical and Biomedical Analysis. 2012;61:191–198.
- Carpenter J. US Pharmacopeia. A review of drug quality in Asia with focus on anti-infectives. Available at: http: pdf.usaid.gov/pdf_docs/PNADH154.pdf. Accessed May 30, 2014.
- NIDQC, Project: Development of analytical methods to detect counterfeit drugs using modern spectroscopic technologies: Raman, Fourier Transform Infrared (FTIR), X-ray Diffraction (XRD), Research Collaboration between NIDQC and USP / PQM.
- Green RL, Brown CD. Raw-material authentication using a handheld Raman spectrometer. Pharm Technol. 2008;32:148, 150, 152, 154, 156, 158, 160, 162.
- Green RL, Brush R, Jalenak W, Brown CD. Verification methods for 198 common raw materials using a handheld Raman spectrometer. Pharm Technol. 2009;33:72–82.
- Gryniewicz-Ruzicka CM, Arzhantsev S, Pelster LN, Westenberger BJ, Buhse LF, Kauffman JF. Multivariate calibration and instrument standardization for the rapid detection of diethylene glycol in glycerin by Raman spectroscopy. Appl Spectrosc. 2011;65:334–341.
- Kauffman JF, Gryniewicz-Ruzicka CM, Arzhantsev S, et al. Pharmaceutical surveillance with rapid spectroscopic screening technologies. Am Pharm Rev. 2010;13:58–62, 64.
- Ahmed MK, McLeod MP, Nezivar J, Giuliani AW. Fourier transform infrared and nearinfrared spectroscopic methods for the detection of toxic diethylene glycol (DEG) contaminant in glycerin based cough syrup. Spectroscopy. 2010;24: 601–608.
- Gryniewicz CM, Spencer JA, Hankins, M, KauffmanJF. Spectroscopic methods for rapid determination of diethylene glycol in glycerin. Am Pharm Rev. 2007;10(7):24-30.