The World Health Organisation (WHO) defines counterfeit medicines as those that have been "deliberately and fraudulently mislabelled according to identity or source'"1. Such illegitimate drugs can have defects in their active pharmaceutical ingredients (APIs), excipients or coating, or their packaging might have been tampered with. The harm resulting from them is unpredictable and can range from treatment ineffectiveness to drug resistance and even death. Additionally, drug counterfeiting is not limited to a class or formulation type and can be encountered anywhere in the wholesale supply chain. In this respect, rapid and mobile techniques that can authenticate medicines where they are encountered are favourable

Assi, Sulaf

Bournemouth University Research Online

Handheld Raman spectroscopy offers this advantage since thesespectrometers are light in weight, have long battery lifetimes and canoperate over a wide temperature range, including both hot and coldclimates2,3. However, one issue that is often encountered withauthenticating medicines using handheld Raman spectroscopy, andmainly attributed to the Raman activity of authenticated medicines, is that drugs with low API contents and high excipient concentrationsoften fluoresce and have weak Raman activity3,4. The latter phenomenonis often encountered when using a 785nm laser, which is used since ithas the advantage of high sensitivity. Fluorescence can be mitigated byusing a further wavelength laser (such as a 1064nm one); however, thisrisks lower sensitivity5. The use of dual laser handheld Raman spectroscopy was successfulin the chemical characterisation and authentication of type 5The World Health Organisation (WHO) defines counterfeit medicines as those that have been “deliberately andfraudulently mislabelled according to identity or source’”1. Such illegitimate drugs can have defects in their activepharmaceutical ingredients (APIs), excipients or coating, or their packaging might have been tampered with. The harm resulting from them is unpredictable and can range from treatment ineffectiveness to drug resistance andeven death. Additionally, drug counterfeiting is not limited to a class or formulation type and can be encounteredanywhere in the wholesale supply chain. In this respect, rapid and mobile techniques that can authenticatemedicines where they are encountered are favourable.IN-DEPTH FOCUS: RAMAN Authenticating medicineswith dual laser handheldRaman spectroscopySulaf AssiBournemouth University12 European Pharmaceutical Review VOLUME 21 ISSUE 6 2016© kasezo / Shutterstock.comphosphodiesterase inhibitors, which containedconcentrations of APIs in the range of 5-15%m/m6. This article describes the results thatwere obtained when handheld Raman was usedto authenticate medicines with low APIcontents and of diverse classes. Experimental designThe medicines investigated in this studyincluded both lifesaving and lifestyle products;these five products were the antihistaminedrug loratadine; antidepressant citalopram; the antihyperlipidemics atorvastatin andsimvastatin; and vardenafil, a sexual stimulant(Table 1). All of these medicines had low dosesof APIs, in the range of 5-15% m/m. Brandedand generic batches of them were obtainedfrom France, India, Lebanon, Turkey, the UK andvia the Internet. Additionally, APIs and the mainexcipients commonly present in the drugs werepurchased from Chemical Suppliers.Raman spectra of medicines were acquiredusing the Bruker BRAVO handheld Ramaninstrument, equipped with dual laser andcharged coupled device detector with thermo -electric cooling. Four spectra were collected from both sides of tablets such that tablets were rotated between measurements. Each spectrum was the sum of one scan over the wavenumber range of300-3200cm-1. Spectra of APIs and excipients were collected throughtransparent glass vials and each vial was shaken and repositioned aftereach measurement.Next, the spectra of APIs, excipients and medicines were exportedin Matlab 2014b where multivariate classification algorithms wereapplied. The two algorithms were: correlation in wavenumber space(CWS) and principal component analysis (PCA). For the CWS algorithm,the test medicine spectrum was comparedagainst the spectra of each of the excipientsand the authentic medicine. In this sense, acorrelation coefficient value (r-value) of ≥ 0.95indicated a match and vice versa. For PCA, the first two scores plots of theauthentic and counterfeit product were compared in relation to the95% equal frequency ellipses. The 95% equal frequency ellipses werecalculated using the authentic scores. In this respect, if an authenticscore was encountered outside the ellipse, this was considered a type Ierror. However, if a counterfeit score was encountered inside theellipse, this was considered a type II error. Results and discussionRaman activity of medicines with low APIsBoth branded and generic versions of each of the five medicines in thestudy were evaluated. All these medicines had API concentrations below15% m/m and this introduced a challenge in the authenticationprocess. Unlike branded medicines, the excipients in generic medicineswere not always known7. Moreover, the Ramanactivity of the individual medicines depends onthe activity of the API as well as the excipients. In general, APIs are Raman-active providedthey are present in a high concentration withinthe product3. Nonetheless, the Raman activityof excipients is often masked by fluorescence3 that impacts the Ramanactivity of the overall medicine. One way of overcoming thefluorescence of excipients is to use sequential shifted excitation (SSE)8.In this work, SSE algorithm was used for authenticating medicines withmultiple excipients and low doses of APIs. The algorithm provedsuccessful in mitigating fluorescence of the medicines, yet had variableeffects on the Raman activity of the medicines where the scatteringintensity was a key factor. Though scattering intensity had arbitraryunits, its value was directly proportional to the concentration(s) ofRaman active substance(s)9. Hence, four parameters were taken into account when evaluatingthe Raman activity of the medicines. These were: fluorescence of theproduct, scattering intensity, range of scattering (over both lasers) andnoise. Where known, the number of excipients present in the medicinesIN-DEPTH FOCUS: RAMAN VOLUME 21 ISSUE 6 2016 European Pharmaceutical Review 13Table 1: Details of the products used in the studyProduct name API Dose (mg) NTP Purchase sourcesAtorvastatin atorvastatin 10 8 France, India,calcium Lebanon,trihydrate Turkey, UKCitalopram citalopram 10 10 Lebanon, UKhydrobromideLoratadine loratadine 10 11 Internet, UKSimvastatin simvastatin 20 Lebanon, UKVardenafil vardenafil 20 30 India, Lebanon,hydrochloride Turkey, Internet, UKFigure 1: Raw Raman spectra of branded products of (a) atorvastatin, (b) citalopram, (c) loratadine, (d) simvastatinand (e) vardenafil, measured using the Bruker BRAVO handheld Raman spectrometer equipped with dual laserRapid and mobile techniques thatcan authenticate medicines where theyare encountered are favourable 14 European Pharmaceutical Review VOLUME 21 ISSUE 6 2016ranged between four and 20 (Table 2). Hence,the multiple constituents present in themedicines affected the Raman activity of them.It is noteworthy to mention that the number ofexcipients did not affect the Raman activity of the medicines (Figure 1; page 13). Hence, allthe evaluated medicines, except for citalopram, had scatteringintensities in the range of 25,000 (observed for atorvastatin) and 60,000arbitrary units (observed for simvastatin). Citalopram had a scattering intensity of 5,000 arbitrary units;however, it showed numerous peaks over the two laser wavenumbers(the first laser up to 2,000 cm-1 and the second between 2,000 and3,000cm-1). The remaining four medicinesshowed scattering mainly in the first laser range (7-12 peaks), and showed only one peak in the second laser range correspond-ing to the CH group. None of the medicinesshowed fluorescence within their spectra.Moreover, all medicines’ spectra showed minimum noise (S/N ratio in the range of 10-40).Spectral evaluation of medicinesIn both laser ranges, the spectra of the medicines showedcorresponding peaks to the API, main excipient and titanium dioxidewhere present (Figures 1 and 2). Corresponding APIs’ peaks showedscattering in the range of 1,000-2,000cm-1 and around 3,000cm-1. The main excipients (being lactose and microcrystalline cellulose)showed scattering over the full wavelength range when titanium dioxidewas not present. Thus, the presence of titanium dioxide masked thesignal between 300 and 700cm-1. In this case, the presence of titaniumdioxide was detected in all products except for loratadine, which was not film coated (Figure 1). This is useful since it enables thethickness of coating to be detected by observing the scattering of titanium dioxide6. Regarding the main excipients, lactose was present in threeproducts: atorvastatin, loratadine and simvastatin (Table 2) with featured peaks around 1,000cm-1 Furthermore, micro-crystalline cellulose was featured in the spectra of citalopram andvardenafil (between 1,000 and 1,500cm-1) as it was their main excipient.The featuring of the main excipient in the spectra of medicines wasuseful in allowing one to compare when the generic medicine con-tained the same main excipient as the branded medicine. This was thecase in this work, where lactose and MCC spectral features were seen inthe spectra of the generic test medicines.Spectral authentication of medicinesSubsequently, the spectra of the test medicines were compared against the authentic medicines, titanium dioxide and the mainexcipients’ spectra. For atorvastatin medicines tested, six productsmatched the spectra of the authentic medicine (r value = > 0.98), andshowed r values in the range of 0.85-0.92 and 0.17-0.32 for lactose Table 2: Constituents of the products used in the studyProduct API Main excipient List of excipientsAtorvastatin atorvastatin lactose calcium carbonate,calcium monohydrate microcrystalline cellulose,trihydrate lactose monohydrate,croscarmellose sodium,polysorbate 80, hydroxypropylcellulose, magnesium stearate,hypromellose, macrogol 8000,titanium dioxide, talc,simethicone emulsionCitalopram citalopram microcrystalline mannitol, microcrystallinehydrobromide cellulose cellulose, colloidal anhydroussilica, magnesium stearate,hypromellose, titanium dioxide,macrogol 6000Loratadine loratadine lactose lactose monohydrate,monohydrate microcrystalline cellulose, maizestarch, magnesium stearateSimvastatin simvastatin lactose butylated hydroxyanisole,monohydrate ascorbic acid, citric acidmonohydrate, microcrystallinecellulose, pregelatinised maizestarch, lactose monohydrate,magnesium stearate,hypromellose, hydroxy propylcellulose, titanium dioxide, talc,iron oxide yellow, iron oxide redVardenafil vardenafil microcrystalline crospovidone, magnesiumhydrochloride cellulose stearate, microcrystallinecellulose, colloidal anhydroussilica, macrogol 400,hypromellose, titanium dioxide, ferric oxide yellow,ferric oxide redAPI: Active pharmaceutical ingredientFigure 2: Raw Raman spectra of branded products of (a) lactose, (b) MCC and (c) TiO2, measured using the Bruker BRAVO handheld Raman spectrometer equipped with dual laserhandheld dual laser Ramanequipped with SSE was successful incharacteristing medicines with lowamounts of APIs IN-DEPTH FOCUS: RAMAN RamanWorkStation tm Raman Analyzer for Research785 nm – 1 Wavelength, 1 Instrument Transmission to Millimeters to Microns•  Raman Microscopy & Imaging•  Transmission Raman (Variable Spot Size)•  P hAT Technology (mm)•  Video Capture & Image Overlay•  Robust, Reliable, Reproducible Raman•  Qualitative & Quantitative AnalysisHTS ScreeningTransmission  BackscatterPolymorphScreeningP hAST  Mapand titanium dioxide, respectively. Oneatorvastatin test medicine failed theidentification as it mismatched the authenticproduct and showed an r value of 0.78 againstthe authentic reference (Figure 3). Theaforementioned test medicine showed r valuesfor lactose and titanium dioxide of 0.69 and0.29, respectively. This indicated that this batchhad the main ingredients listed but in differentamounts than the authentic reference. Figure 4 (page 16) shows the correlationmap of test atorvastatin tablets against thereference medicine, lactose and titaniumdioxide. A high match (ir values of 0.95-1) isdisplayed in a dark red colour, whereas a highdissimilarity (r values of around 0) is shown indark blue. In this respect, authentic batches(columns and rows 13-50) showed r values ofabove 0.98. On the other hand, the counterfeitbatch (columns and rows 51-60) showed r values in the range of 0.77-0.79 against theauthentic reference. Similarly, mismatches were also encount -ered when authenticating vardenafil products.In this respect, authentic vardenafil batches gave r values in the rangeof 0.96-0.98, 0.46-0.58 and 0.70-0.92 against the reference medicine,lactose and titanium dioxide. Conversely, counterfeit vardenafil batchesgave r values in the range of 0.78-0.92, 0.13-0.83 and 0.38-0.95 againstthe reference medicine, lactose and titanium dioxide. This indicated thediversity and inconsistency of ingredients present in the counterfeitbatches evaluated. On the other hand, no mismatches were observed for citalopram,IN-DEPTH FOCUS: RAMAN Figure 3: Raw Raman spectra of authentic (blue) counterfeit (red) atorvastatin products measured using the BrukerBRAVO handheld Raman spectrometer equipped with dual laserloratadine and simvastatin medicinesevaluated. Accordingly, the tencitalopram medicines evaluated gave r values of above 0.99 for the citalopramreference medicine. Additionally, theygave r values in the range of 0.72-0.92and 0.3-0.41 for titanium dioxide andmicrocrystalline cellulose, respectively.The identical match between the afore -mentioned ten batches and theauthentic reference could indicate theywere from the same manufacturingsource as the authentic product.Likewise, loratadine and simvastatinmedicines gave r values of above 0.96against their authentic references.Additionally, loratadine gave r values inthe ranges of 0.93-0.95 and 0.01-0.012 forlactose and titanium dioxide. This was because loratadine medicineshad high concentrations of lactose in their formulations and notitanium dioxide. On the other hand, simvastatin medicines showed r values in the ranges of 0.48-0.63 and 0.70-0.84 for lactose and titaniumdioxide, which corresponded to the excipient content and coating. To confirm the reliability of the correlation method in authenticatingthe aforementioned medicines, PCA was applied to the spectra ofauthentic and counterfeit batches. For most medicines, PCA was able toconfirm the correlation results with no type I or type II errors. Type Ierrors were only encountered when authenticating atorvastatin andloratadine medicines (Figure 5). Two authentic atorvastatin batches and seven authentic loratadine batches were misclassified using the95% equal frequency ellipse. This could be attributed to the diversity ofmatrices among the medicines. Subsequently, future work should lookinto a more quantitative approach for APIs and main excipients inmedicines with diverse matrices.ConclusionIn summary, handheld dual laser Raman equipped with SSE wassuccessful in characteristing medicines with low amounts of APIs. The SSE algorithm showed to be successful in mitigating thefluorescence encountered when multiple excipients were present in the formulation. Additionally, correlation and the PCA method weresuccessful in authenticating the majority of the products. Few productsshowed type I errors when classified using PCA. Thus, future work willinvolve the development of quantitative models for authenticatingbranded and generic medicines.16 European Pharmaceutical Review VOLUME 21 ISSUE 6 2016IN-DEPTH FOCUS: RAMAN 1. WHOFactsheetN˚275. Medicines: spurious/falsely-labelled/ falsified/counterfeit (SFFC)medicines 2012. Available from: http://www.who.int/mediacentre/factsheets/fs275/en/2. S Assi. Laboratory based versus handheld instruments: What you gain and what you lose.American Pharmaceutical Review. 20123. S Assi. Investigating the quality of medicines using handheld Raman spectroscopy.European Pharmaceutical Review. 2014; 19 (5), 55-604. MR Witkowski. The use of Raman spectroscopy in the detection of counterfeit andadulterated pharmaceutical products. American Pharmaceutical Review. 2005; 8, 56-625. S Assi. Raw material identification using dual laser handheld Raman spectroscopy,European Pharmaceutical Review. 2013; 18 (5), 25-316. S Assi. Identification of Counterfeit Drugs using dual laser handheld Ramanspectroscopy. European Pharmaceutical Review. October 2015; Issue 67. SS Dunne and CP Dunne. What do people really think of generic medicines? A systematicreview and critical appraisal of literature on stakeholder perceptions of generic drugs.BMC medicine. 2015; 13(1), p.18. JB Cooper, M Abdelkader and KL Wise. Sequentially shifted excitation Ramanspectroscopy: novel algorithm and instrumentation for fluorescence-free Ramanspectroscopy in spectral space. Applied spectroscopy. 2013; 67(8), pp.973-9849. P Venezuela, M Lazzeri and F Mauri. Theory of double-resonant Raman spectra ingraphene: intensity and line shape of defect-induced and two-phonon bands. PhysicalReview B. 2011; 84(3), p.035433ReferencesDr Sulaf Assi is a lecturer in forensic science andProgramme Leader for Forensic Investigation at the Facultyof Science and Technology, Bournemouth University (BU),UK, where she has worked since 2013. She obtained aBachelor of Pharmacy from the Faculty of Pharmacy, BeirutArab University, Lebanon, before becoming a hospitalpharmacist and pursuing her MSc in pharmacy. After this,Sulaf did her PhD in pharmaceutical analysis at the School of Pharmacy,University of London, UK. Her research expertise is in: counterfeitmedicines; drug abuse and misuse; patient safety; clinical pharmacy;analytical techniques; and multivariate data analysis.Figure 4: Correlation map of the Raman spectra of (1) titanium dioxide, (2) lactose, (3-50) authentic atorvastatin and (51-60) counterfeit atorvastatin. The colour map onthe right shows the corresponding range to the r values. A perfect match (r = 1) isdisplayed in dark red, whereas a perfect mismatch (r = negative) is displayed in dark blueFigure 5: PCA scores plot of the Raman spectra authentic (blue) and counterfeit (red) of (a) atorvastatin and (b) loratadinemedicines with the 95% equal frequency ellipses plotted around the authentic scores

Authenticating medicines with dual laser handheld Raman spectroscopy

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