Raman spectroscopy and hierarchical cluster analysis for the ingredients characterization in different formulations of paracetamol and counterfeit paracetamol

Vibrational Spectroscopy 102 (2019) 112–115

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Raman spectroscopy and hierarchical cluster analysis for the ingredients characterization in different formulations of paracetamol and counterfeit paracetamol

T

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Hina Alia, , Rahat Ullaha, Saranjam Khanb, Muhammad Bilala a b

Agri. & Biophotonics Division, National Institute of Lasers and Optronics (NILOP), Nilore, Lehtarar Road, Islamabad, Pakistan Islamia College, Peshawar, Pakistan

A R T I C LE I N FO

A B S T R A C T

Keywords: Panadol Acetaminophen Caffeine Excipients Spectroscopy HCA Optizorb® technology

The distribution of pharmaceutical ingredients in tablet influences its effectiveness. Direct analysis of tablet using Raman spectroscopy generates information on internal distribution of tablet ingredients. Raman spectra of local and imported formulations of paracetamol clearly discriminated signatures of paracetamol and caffeine in Panadol and Panadol Extra respectively in intact, sliced and powdered tablets/caplets. The presence of few excipients like titanium dioxide in surface coating and calcium carbonate was also detectable in imported Panadol Advance and Panadol Extra caplets with Optizorb® technology which helps in quick disintegration of tablet inside body. Moreover, locally available counterfeit paracetamol was also analysed that showed less intense peaks for active ingredient i.e. paracetamol and few enhanced peaks for excipients as separated by hierarchical cluster analysis (HCA). It is suggested that Raman spectroscopy alongwith chemometrics can be effectively used for the rapid identification and distribution of active drug ingredients in tablets for quality control and detection of counterfeit drugs.

1. Introduction Acetaminophen is the generic form of paracetamol which is widely used as an analgesic and antipyretic available with different brand names. Self-medication for the relief of pain and fever is very common by taking this over-the-counter (OTC) medicine. The active ingredient (paracetamol) is 500 mg per tablet and the maximum suggested dose is 4 g/day. Over dosage of paracetamol can cause liver damage, moreover its daily use for long period of time has been reported to cause renal damage and blockage in synthesis of DNA replication [1]. Paracetamol is also available in combination with caffeine. Caffeine is an agrochemical, therapeutic and physcoactive agent and is present in number of dietary sources [2]. One dose of paracetamol (2 tablets) contains 130 mg caffeine more than a cup of tea and nearly equivalent to 8 oz. of coffee. Recommended dose (520 mg/day) of caffeine should not increase due to dietary intake of caffeinated drinks [3]. Pharmaceutical properties and bioavailability of a drug depends on its molecular structure. Paracetamol crystalizes in three different isoforms. Form I is thermodynamically more stable and of commercial interest due to mixing with binders in the tablet form. The other two forms are Form II that is metastable orthorhombic crystals and Form III,

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which is very unstable. Form II is used in direct compression of tablets and has more solubility [4]. The main difference between different pharmaceutical formulations of paracetamol is the quick absorption and efficacy in the body. Drug companies use different technologies or add extra ingredients in medicines to increase the efficacy and absorption of medicine in stomach and eventually in blood like addition of caffeine in Panadol extra for efficacy. Panadol is a registered trademark of the GlaxoSmithKline (GSK) group of companies, different formulation of paracetamol have been manufactured by GSK and available throughout the world. Another recently introduced patented technology by GSK is Panadol Optizorb® includes ingredients that help in quick disintegration of tablet in five minutes and effective in short time as compared to standard Panadol [5]. In addition to ingredient characterization, the counterfeit medicines should also be identified as it’s a profitable business but poses a serious public health issues especially in low income nations. Different analytical techniques like chromatographic, electrophoretic, electrochemical and spectroscopic have been established in pharmacopoeias for the analysis of drugs [6]. However, Spectroscopic techniques i.e. Raman, Near Infra Red, visible and UV absorption are quick, less laborious and have been used for qualitative and

Corresponding author. E-mail address: [email protected] (H. Ali).

https://doi.org/10.1016/j.vibspec.2019.05.002 Received 5 January 2019; Received in revised form 26 April 2019; Accepted 3 May 2019 Available online 08 May 2019 0924-2031/ © 2019 Elsevier B.V. All rights reserved.

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recording data multiple times from different tablets and batches. The intensity of Raman data reflects the active ingredient, packing density of sample and its homogeneity. The Raman peaks recorded in the region from 1420 -1612 cm−1 attributed to C–N amide and CeC aromatic stretching modes and were observed in all Panadol tablets/caplets (Fig. 1) taken from whole intact tablet surface. These peaks are specific to monoclinic Form I of paracetamol [1], thus confirming the presence of monoclinic Form I. The difference between standard Panadol and Panadol Extra lied in the region 440-740 cm−1 that corresponds to the caffeine (Table 1). The peak intensity was high on the surface and inside of local PE tablet i.e. a strong feature at 553 cm-1 and assigned to the CNC and O = CN deformations in the pyrimidine ring of caffeine [11,12]. The intensity of this peak dropped down in powdered local PE as compared to the surface spectrum (Figs. 1 and 3). The variation in relative intensities for this peak were observed in local PE from surface, sliced tablet and powdered spectra showing unequal distribution of caffeine whereas slight difference in intensity was observed for imported PE (Figs. 1–3). There are few additional peaks in the surface (intact caplet) spectra of PAI and PEI at 513 cm−1, hump at 626 cm−1 and 1085 cm-1 that were absent in the spectra of the same brand available in Pakistan. Peak at 513 cm-1 and a clear hump from 626 to 636 cm-1 were assigned to Titanium dioxide (TiO2), while 1085 cm-1 corresponds to calcium carbonate [13]. TiO2 is a naturally occurring mineral and is one of the excipients in tablet coating of PAI and PEI caplets, it act as a opacifier and coating agent in tablets and has other pharmaceutical uses [14]. Anatase is one of the three mineral polyforms of TiO2 and has defined peaks at 513 and 636 cm −1 (Fig. 1). All the Panadol tablets showed peak at 626 cm −1 assigned to Amide IV band (HeNeC deformation); this peak merges with TiO2 bending vibration of anatase at 636 cm −1 thus forming a broad band ranging from 626 to 636 cm −1. The absence of these two bands (513 and 636 cm−1) in powdered and sliced caplets of PAI and PEI confirmed their presence in caplet surface coating only. Another distinctive spectral peak was observed at 1083 cm −1 in PAI and PEI which remained intact even in the spectra recorded from sliced caplets and powder; this ingredient was found to be calcium carbonate. It is used as coating and buffering agent and is reported to have a strong peak at 1085 cm−1 associated with symmetric stretching (ν1 vibration) of carbonate (CO3) [13,15]. Dissolution of PAI and PEI caplets is enhanced in the presence of carbon dioxide released from calcium carbonate and formation of calcium alginate when calcium ions react with alginic acid (excipient of Panadol) in the stomach [16]. The spectra of counterfeit paracetamol intact tablets were compared with branded Calpol and Panadol (Fig. 4), the intensity of major peaks for acetaminophen showed decreased intensity in counterfeit paracetamol. Other enhanced Raman and noisy peaks at 253, 523, 690, 742,

quantitative analysis of paracetamol and its polyforms [7,8]. Molecular fingerprinting of chemical compounds can be easily done with the help of Raman spectroscopy and has been widely used in pharmaceutical industry [9]. The present study was planned to characterize and compare ingredients among intact paracetamol tablets, sliced and powdered tablets by using Raman spectroscopy. Moreover counterfeit OTC medicine is becoming an alarming issue and paracetamol is available in local market in bulk without packaging. To fingerprint the counterfeit paracetamol tablets, its Raman spectra were also compared with the branded one. 2. Materials and methods Panadol is a registered trademark of Glaxo SmithKline (GSK). In this study Calpol (C), Panadol (P) and Panadol Extra local (PEL) from GSK, Pakistan in tablet form and Panadol Advance Ireland (PAI), and Panadol Extra Ireland (PEI) from GSK, Ireland with Optizorb® technology in caplet form were compared. These commercially available tablets contain 500 mg of paracetamol as Panadol, Calpol and Panadol Advance whereas addition of 65 mg caffeine as Panadol extra. In addition to these, the counterfeit paracetamol tablets were also collected from local dispensary for analysis. Raman spectra were recorded using PeakSeeker PRO-785 coupled with microscope RSM-785, Agiltron USA) having a spectral resolution of 10 cm−1. It employed diode laser emitting at 785 nm for sample excitation. The incident laser power was 10 mW with an acquisition time of 10 s. The Raman data was recorded from surface layer, powdered form and sliced tablet/caplet. The tablet/caplet was focused on one area with the help of microscope and the data was recorded randomly from different areas. The spectra were recorded multiple times with different tablets from two different batches with spectral region of 200-1800 cm−1. MATLAB software (release 2009a) [10] was employed for Raman band deconvolution and data pre-processing (smoothening, background adjustment and normalization). Furthermore normalized Raman data were treated using chemometric method i.e. HCA using the group average linkage method for cluster analysis using Origin Pro 2017 software. 3. Results and discussion The average Raman spectra from surface, inner sliced and powdered tablet of different paracetamol formulations are illustrated in Figs. 1–3. All the formulations share similar bands for acetaminophen except for few peaks of caffeine in Panadol extra and ingredients added in Optizorb® technology. The reproducibility of the results was confirmed by

Fig. 1. Normalized Raman spectra of different formulations of Paracetamol from tablet surface. 113

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Fig. 2. Normalized Raman spectra of different formulations of Paracetamol from inner side of sliced tablet.

Fig. 3. Normalized Raman spectra of different formulations of Paracetamol from powdered tablet. Table 1 Distinctive Raman peaks data from surface, sliced tablet and powdered form in different Panadol formulations. S. no.

Peak position

1 2 3

442 482 513

4

555

5

626

6 7

626-636 738

8

1085

Peak assignment

Calpol (local)

Panadol (local)

Panadol Extra (local)

Panadol Advance (Ireland)

Panadol Extra (Ireland)

Wavenumber (cm−1) δ(pyrimidine ring) + δ(CNO) + δ(CH) δ(pyrimidine ring) + δ(CNO) + δ(CH) symmetric ν(TiO2) stretching vibration for anatase assigned to the CNC and O = CN deformations in the pyrimidine ring (Caffeine) Amide IV band (H–N–C deformation) δ(TiO2) bending vibration for anatase δ(pyrimidine, imidazole ring) + δ(CH3) + ρ(CH3) symmetric stretching (ν1 vibration) of carbonate (CO3) Calcium carbonate

S – – –

I – – –

P – – –

S – – –

I – – –

P – – –

S + + –

I – + –

P – – –

S – – +

I – – –

P – – –

S + + +

I – + –

P – + –

–

–

–

–

–

–

+

+

+

–

–

–

+

+

+

+

+

+

+

+

+

+

+

+

–

+

+

–

+

+

– –

– –

– –

– –

– –

– –

– +

– +

– +

+ –

– –

– –

+ +

– +

– +

–

–

–

–

–

–

–

–

–

+

+

+

+

+

+

S = Surface, I = Sliced tablet, P = Powdered, + = presence of peak, - = Absence of peak.

886, 1696, 1774 and 1857 cm −1 positions showed increased and substandard quality of excipients in counterfeit paracetamol. Furthermore HCA was applied on Raman data that converts data points in reduced dimensions by extracting dominant patterns in complex matrics [17]. The results revealed the differences among apparently similar spectra by using average group linkage method. The dendrogram (Fig. 5) showed 3 main clusters with 7 subclusters. Cluster

1 showed the separation of caplet of imported formulations (PEI and PAI) in the subcluster as 1S and 2S due to the presence of additional peaks of TiO2 and CO3. Subcluster 6 grouped together powdered form of PEI and PEL (2 P and 3 P) due to the presence of caffeine peaks and separated from rest of the paracetamol tablets. The last and distant cluster belongs to counterfeit paracetamol (F) that clearly separated out from rest of the paracetamol formulations. The hierarchical tree showed 114

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Fig. 4. Normalized Raman spectra of counterfeit Paracetamol in coparison with branded forms.

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Fig. 5. Dendrogram showing cluster analysis of different formulations of paracetamol from intact tablet i.e. Surface (S), Sliced (I) Powdered (P) form and F(counterfeit paracetamol).

that Raman spectra of both intact tablet and its powdered forms were well differentiated based on ingredients. The clusters developed can be effectively used to distinguish different formulations of paracetamol and its imitation based on Raman data. In this study comparison of different formulations of paracetamol by Raman spectroscopy showed that imported Panadol Advance and Panadol Extra have smooth and homogenous distribution of ingredients as compared to the local ones. Moreover other excipients like caffeine, calcium carbonate and titanium dioxide also showed distinctive peaks that separated standard Panadol from Panadol Extra with optizorb technology. To the best of our knowledge, the fingerprint of calcium carbonate in optizorb technology which helps in quick disintegration and mild effect on stomach has not been reported previously. This proves the potential of Raman spectroscopy alongwith chemometrics for determining the distribution of active ingredients and composition of different tablets for quality control during and after production.

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