Kelani 2020

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H. Bebawi, M. S. ElSherbiny and S. M. Eid, Anal. Methods, 2020, DOI: 10.1039/D0AY01749C.
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TECHNICAL NOTE
Kássio M. G. Lima et al.
ATR-FTIR spectroscopy with chemometric algorithms of multi-
shall the Royal Society of Chemistry be held responsible for any errors
variate classifi cation in the discrimination between healthy vs.
dengue vs. chikungunya vs. zika clinical samples
or omissions in this Accepted Manuscript or any consequences arising
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Page 1 of 38 Analytical Methods
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FTIR combined with chemometric tools (Fingerprinting spectroscopy) in
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comparison to HPLC; Which strategy offers more opportunities as a green
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analytical chemistry technique for the pharmaceutical analysis
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Khadiga M. Kelani a,b, Mamdouh R. Rezk a, Hany H. Monir a, Menna S.
Analytical Methods Accepted Manuscript

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14 ElSherbiny b, Sherif M. Eid c *
Published on 16 November 2020. Downloaded by Carleton University on 11/28/2020 11:12:53 AM.
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a Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, El-
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20 Kasr El-Aini Street, ET-11562 Cairo, Egypt.
21 b Analytical
22 chemistry department, Faculty of Pharmacy, Modern University for
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24 technology and information, Cairo, Egypt.
25 c Analytical
26 chemistry department, Faculty of Pharmacy, October 6 University, 6
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28 October City, Giza, Egypt.
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35 Corresponding Authors
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37 * Email: Sheriefmohammed@o6u.edu.eg
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39 : sherief055@icloud.com
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44 Keywords:
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46 GAC; ATR-FTIR; PLSR chemometrics; HPLC; Ketoprofen and hyoscine;
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48 Benzocaine and dextromethorphan HBr
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52 Conflict of interest:
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54 The authors declare no conflict of interest.
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Analytical Methods Page 2 of 38
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Abstract: View Article Online
DOI: 10.1039/D0AY01749C
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Fourier transform infrared spectroscopy (FTIR) is a widespread technique
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that can provide a chemical signature (fingerprints) of either solid, liquid, or gas
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samples with a wide range of analytical applications. High-performance liquid
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chromatography (HPLC) is a leading analytical strategy for pharmaceutical

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14 analysis. Here we present a side-by-side comparison of the potential of these
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16 techniques for quantitative analysis of pharmaceutical active ingredient
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18 combinations in the light of the green analytical chemistry (GAC) principles. The
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20 methods were successfully applied for the analysis of Ketoprofen (KTP)/
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22 hyoscine (HYS) and Benzocaine (BENZ)/ dextromethorphan HBr (DEX) in their
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24 binary drug mixtures and pharmaceutical preparations. In the FTIR analysis,
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26 calibration models were constructed based on partial least squares regression
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28 (PLSR) with satisfactory regression coefficients (r2) (0.9998, 0.9994, 0.9855, and
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30 0.9895) for KTP, HYS, DEX, and BENZ, respectively, over a wide linearity
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32 range (10-100, 10-100, 5-75, and 10-100 µg/ml) that covers the concentration
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34 ratios in the market samples. External validation using validation set and Internal
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36 validation using leave-out-one-cross-validation calculations were performed, and
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38 small root-mean-square-error-of-cross-validation (RMSECV) values were
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40 obtained indicating good resolving power of the models. The same performance
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42 was obtained using the HPLC method for separation of the same mixtures with
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44 (r2) equals (0.9998, 0.9999, 0.9998, and 0.9998) over a linear range of (50-1000,
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46 10-200, 5-100, 5-100 µg/ml) for KTP, HYS, DEX, and BENZ, respectively. The
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48 HPLC methods were validated following ICH guidelines with good recovery
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50 percent in ranges of 98 – 100 % were obtained. The statistical comparison for the
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52 FTIR and HPLC methods of the analysis showed almost the same results with
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54 good applicability towards the commercial dosage forms. Concerning the GAC
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56 twelve principles, a detailed comparison was performed to highlight the
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58 opportunities of each technique. FTIR-PLSR analysis showed superior
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60 performance as it allows for less solvent consumption, portability, less generated
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waste, short operation time, less operation cost, less energy consumption, more
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operator safety, and it is easily coupled with chemometric tools. Besides, FTIR is
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a direct analytical technique that can be used for the analysis of samples in all the
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physical forms (solid, liquid, and gas) without modifications.
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1. Introduction View Article Online
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The quality of pharmaceutical products is of vital importance for the
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patients’ safety. Although chromatographic techniques have a long history of
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active pharmaceutical analysis (APA), this work demonstrates the opportunities
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offered by the vibrational spectroscopic methods for APA in comparison to the

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14 traditional HPLC methods in the light of the 12 objectives of green analytical
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16 chemistry (GAC) (1).
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18 The idea of GAC was introduced by Anastas and Warner in 1999 (2)to
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20 remove the side effects of the analytical chemistry labs on the environment and
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22 the hazards that may affect human health. This idea was very attractive to many
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24 chemists, especially that following the guidelines of GAC is cheaper than
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26 addressing health problems or purifying the polluted environment (3, 4).
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28 GC, HPLC, NMR, MS, UV/VIS, and electrochemical methods are widely
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30 used for the analysis and quality control of pharmaceutical products during all the
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32 stages of manufacture from raw materials to finished products (5). Analysts are
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34 constantly trying to make all these techniques as green as possible in all stages of
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36 the analysis, but they are usually faced with several obstacles. For example, the
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38 widely used chromatographic methods as HPLC has many side effects and
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40 drawbacks that prevent this technique from being full green analytical techniques
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42 as the hazardous mobile phase components which are mostly organic solvents.
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44 These organic solvents are usually volatile; therefore, they can easily evaporate
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46 and harm the operator and the environment. Also, these techniques are not
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48 portable, expensive, generate a high amount of waste, consume more energy, and
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50 need more time to get results (6).
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52 Accordingly, there is a great responsibility for the analysts and researchers
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54 to find a suitable pathway to minimize the negative effect of the analytical
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56 practices on the environment and the operators. It is a huge challenge for them to
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58 achieve the balance between obtaining high-quality results and improving the
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60 environmental friendliness of the analytical practices at the same time.
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Over the past decade, spectroscopic techniques such as Fourier DOI:
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infrared spectroscopy (FTIR) have been considerably developed in terms of
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instrumentation such as attenuated total reflectance (ATR) unit, sensitivity, and
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software (7). This can be proved by its different applications in the analysis of
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multicomponent market samples (8), doping drugs (9), determination of enzyme

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14 activity (10), biological samples, biomarkers, oils, gases, and cancer cells (7).
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16 FTIR technique has many advantages as being a non-destructive technique
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18 permitting the reuse of samples, analyzing the under-investigation samples in any
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20 physical state without any sample pretreatment either gases, liquids, or solids.
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22 FTIR is a high throughput technique as the IR covers a wide spectral range
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24 (wavenumbers) and the samples can be analyzed at any level (micro-levels or
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26 macro-levels) (11).
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28 With the increasing concern about climate changes, environmental safety,
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30 operator’s safety, and the high demand for cheaper green analytical techniques
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32 that can produce fast high-quality results permitting practically real-time answers,
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34 FTIR spectroscopy seems to offer the most promising option.
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36 However, all of these advantages cannot be completely achieved without
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38 the integration with smart chemometric tools such as partial least squares
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40 regression modelling, that enable the selection and/or the design of the ideal
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42 measurement methods and experiments and offer the largest chemical
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44 information from the spectral data (12).
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46 In the current study, two partial least squares chemometric methods
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48 coupled with ATR-FTIR analysis were developed for the quantitative
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50 determination of mixtures of Ketoprofen/ hyoscine (KTP/HYS) and mixtures of
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52 Benzocaine/ dextromethorphan HBr (DEX/BENZ) in their commercial dosage
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54 forms. Also, two HPLC methods have been optimized and validated for the
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56 quantitative determination of the same mixtures. The combination of KTP and
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58 HYS is prescribed for the fast relief of severe abdominal pain in the renal system,
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60 hepatobiliary system, or gastrointestinal tract. While DEX/BENZ is prescribed
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for the temporary relief of sore throat, sore mouth, pain associated with canker
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sores, and minor irritation. The performance of the ATR-FTIR procedures was
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compared to the performance of the HPLC procedures for the quantification of
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these mixtures in the light of the twelve principles of GAC on the analysis of
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pharmaceutical preparation.

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16 2. Experimental procedures
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18 2.1. Materials and reagents
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20 Ketoprofen (KTP), hyoscine butylbromide (HYS), benzocaine (BENZ),
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22 dextromethorphan HBr (DEX), methanol, acetonitrile, potassium dihydrogen
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24 phosphate, orthophosphoric acid, arginine, benzyl alcohol, propylene glycol,
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26 sodium Bicarbonate, and the anhydrous citric acid were obtained from Sigma-
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28 Aldrich Corporation, GmbH, Germany. Bi-distilled deionized water was
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30 prepared by a Milli-Q® integrated water purification system.
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32 Spasmofen® ampoules were obtained from local pharmacies in Cairo,
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34 Egypt. Each ampoule was labeled to include 100 mg ketoprofen /2 mL and 20 mg
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36 hyoscine butylbromide / 2 mL.
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38 Cēpacol® Extra strength lozenges were purchased from local pharmacies
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40 in Arizona state, USA. Each lozenge was labeled to contain 7.5 mg benzocaine
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42 and 5 mg dextromethorphan HBr.
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46 2.2. Instruments
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48 A Shimadzu Fourier-transform infrared (FT-IR) instrument model
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50 (IRaffinity-1, Japan) with a ceramic light source of high energy, KBr beam
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52 splitter coated with germanium, and a DLATGS detector. The device is equipped
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54 with attenuated total reflectance accessory model 8200HA (PIKE Tech, USA)
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56 which holds a yellow durable hard trapezoid Zinc Selenide (ZnSe) crystal with
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58 the following dimensions (4 mm thickness, 10 mm width, and 80 mm length).
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60 For each sample, three spectra were recorded.
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Waters 2690 Alliance HPLC system including the following: a pump with
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6 a low-pressure mixing system, vacuum degasser, a Waters 996 photodiode array
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8 detector, an auto-sampler with a sample loop of 100 μl and a capacity of 120
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10 vials, a stationary phase composed of C18 Thermo column with the following
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12 diameters (5 μm, 25 cm × 4.6 mm I.D.). The device is equipped with an

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14 empower software that is used for data acquisition and manipulation.
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18 2.3. Standard solutions
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20 A 1 mg mL-1 standard solution for each of the drugs (KTP, HYS, DEX,
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22 and BENZ) was prepared. A weight of 0.05 g from each powder was transferred
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24 into different volumetric flasks. Each flask was completed to 50 ml using
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26 methanol as a solvent.
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30 2.4. FTIR chemometric method
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32 2.4.1. The partial factorial design of the PLSR model
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34 The design of experiments is the key step to increase the chances of
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36 obtaining representative data with useful information. A representative
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38 experimental design was constructed to cover a wide concentration range by
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40 using the multilevel partial factorial design (13). For each drug combination, the
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42 experimental set was designed using two factors (the two drugs found in each
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44 combination) with five concentration levels each (I 5). This requires at least I2
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experiments (52 = 25 mixtures) for each binary pharmaceutical combination. Two
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mixture sets were prepared for each drug combination (HYS / KTP and DEX/
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BENZ mixtures). Each experimental set contains 25 drug mixtures that were
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prepared by transferring the appropriate amounts from the standard solutions into
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different volumetric flasks. Then the volumes were completed to 10 mL using
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methanol.
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mixtures were assigned randomly for the calibration set using the following
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concentrations ranges: 5 – 75 µg mL-1 for HYS and 10 – 100 µg mL-1 for KTP.
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For the second drug-mixture (BENZ and DEX), another 15 laboratory prepared
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mixtures were selected randomly for the calibration set including the following

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14 concentrations: 100 – 1000 µg mL-1 for each drug as shown in table (1). The
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16 mixtures were prepared into a series of 10 mL volumetric flasks using methanol
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18 as a solvent.
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20 Then two validation sets were selected, one for each binary mixture. Each
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22 set consisted of ten mixtures, selected randomly using the remaining
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24 concentrations of the experimental set as shown in table (1).
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28 2.4.2. Spectral data recording steps
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30 A Shimadzu FTIR device has been used for data collection in the mid-
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32 infrared region in the range of 4600 to 400 cm-1. The device parameters were
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34 adjusted in the controlling software (IRsolution version 1.6) with the scanning
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36 resolution equals 4 cm-1 using a SqrTriangle function of apodization in the MID-
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38 IR region. For each mixture; 45 scans were performed in one minute and each
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40 spectrum can give 2180 data points which were obtained on 1.93 wavenumber
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42 intervals.
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44 To obtain a representative FTIR spectrum for each mixture, a background
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46 spectrum was recorded using methanol to eliminate any possible interference of
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48 methanol as a solvent. Then, about 20 L from each mixture was injected on the
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50 surface of the ZnSe prism (ATR unit) to be recorded against the recorded
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52 background. The ZnSe prism cleaned several times using pure methanol between
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54 measurements.
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The pre-processing of recorded spectra is a critical step of the chemometric
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model building, which targets eliminating the undesirable variations
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(instrumental artifacts) and thereby focusing on the important representative
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regions. This is usually done before PLSR calculations. Single or combined pre-

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14 processing techniques (14) (such as baseline correction, detrending,
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16 normalization, Savitzky-Golay derivative algorithm, SNV, and smoothing) were
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18 tested to choose the optimum pre-processing steps. The simple auto-zero baseline
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20 correction was found to be the most suitable pre-processing technique. For
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22 selecting the ideal wavenumber regions for the model calibration and application,
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24 different FTIR spectra regions with diverse start and end were examined. The
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26 spectral regions which gave the smallest values of root mean square error of
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28 calibration (RMSEC) and the largest values of correlation coefficients (R2) were
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30 nominated for constructing their PLSR calibration models.
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32 Finally, for HYS and KTP mixtures, the regions between 4200 - 2500 and
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34 1850 - 400 cm-1 were selected for the building of the PLSR model using The
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36 Unscrambler X version 10.5 (CAMO Software, 2013). Also, for BENZ and DEX
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38 mixtures, the regions between 4200 - 2600 and 1800 - 400 cm-1 were selected for
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40 the building of the PLSR model using the same software.
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44 2.4.4. Effect of the excipients
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46 A test set of ten mixtures was prepared by the under-investigation
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48 compounds using concentration ratios equivalent to the validation set but in the
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50 presence of the expected excipients. The excipients used in Spasmofen®
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52 ampoules such as benzyl alcohol, propylene glycol, and anhydrous citric acid
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54 were prepared in the presence of HYS and KTP at concentrations of 0.02% (w/v)
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56 each. The mixtures were filtered then FTIR analysis was performed using the
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58 same method mentioned earlier for the main drugs.
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Also, the excipients used in Cēpacol® Extra strength lozenges
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FD&C red no. 40, FD&C blue no. 1, Isomalt, flavors, sucralose were prepared in
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presence of BENZ and DEX at concentrations of 0.02% (w/v) each. The mixtures
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were filtered then FTIR analysis was performed using the same method
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mentioned earlier for the main drugs.

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16 2.5. HPLC method
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18 2.5.1. Chromatographic conditions
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20 A Waters Alliance HPLC device model 2690 equipped with a photodiode
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22 array detector, was used for the chromatographic separation. The separation was
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24 done on the C18 thermo column: (5 μm, 25 cm × 4.6 mm I.D.) using an isocratic
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26 mobile phase consisting of acetonitrile and 0.02 M sodium dihydrogen phosphate
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28 at different ratios for both drug-mixtures. The flow rate was 1 mL min-1 for both
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30 drug-mixtures. For HYS / KTP mixture, the mobile phase ratio was acetonitrile:
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32 0.02 M sodium dihydrogen phosphate buffer as 50: 50 v/v and the detection
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34 wavenumber was 220 nm. While for BENZ / DEX mixture, the mobile phase was
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36 acetonitrile: 0.02 M sodium dihydrogen phosphate in a ratio of 40: 60 v/v and
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38 detected at 230 nm.
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42 2.5.2. Construction of the Calibration curves
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44 The HPLC method was calibrated and validated following the ICH
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46 guidelines (15). Aliquots from the stock solutions of HYS (100 to 200 ug ml-1)
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48 and aliquots from the stock solutions of KTP (50 to 1000 ug ml-1) were transferred
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50 separately into a series of 10 ml volumetric flasks. Then, their volumes were
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52 completed to the mark by the mobile phase.
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54 For the second mixtures, aliquots from the stock solution of BENZ
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56 (equivalent to 5 to 100 μg ml-1) and others from the stock solution of DEX
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58 (equivalent to 5 to 100 μg ml-1), were transferred separately into a series of 10 ml
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volumetric flasks, then the volumes were completed to the mark with the mobile
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phase.
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Further, a volume of 20 μl was accurately taken from each flask and
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chromatographed. The obtained peak areas were plotted against their
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concentrations for the construction of the calibration curves and determination of

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14 the recovery percent.
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18 2.6. Application to the pharmaceutical dosage forms
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20 2.6.1. Using FTIR coupled with PLSR Method
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22 Different Spasmofen® ampoules labeled to contain 100 mg ketoprofen /2
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24 mL and 20 mg hyoscine butylbromide / 2 mL were emptied and a stock solution
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26 was prepared. Appropriate volumes were taken to get three different dilutions
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28 (5:25, 10:50, 20:100 μg mL-1 for HYS and KTP respectively) and the volumes
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30 were made to the mark using methanol. Then 20 μl from each volumetric flask
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32 was placed over the surface of ZnSe prism (ATR unit) to be scanned and the
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34 PLSR modelling was performed.
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36 For the second mixture, ten Cēpacol® Extra strength lozenges were ground
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38 thoroughly and weighed accurately. An amount of the powder corresponding to
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40 the average weight of one lozenge was then transferred accurately into a 100 mL
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42 beaker. Only 50 mL of methanol was added and the solution was sonicated then
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44 filtered quantitatively into a 100 mL volumetric flask. The residue was washed
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46 several times with 10 mL aliquots of methanol and then the flasks were completed
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48 to the mark with the same solvent. Also, three different dilutions were prepared
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50 (75:50, 37.5:25, 30:20 μg mL-1 for BENZ and DEX, respectively). Then 20 μl
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52 from each flask was injected over the surface of the ZnSe prism to be scanned
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54 and the PLSR modelling was performed.
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About 10 μl aliquots of the previously prepared ampoule dilutions
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and KTP were chromatographed under the specified chromatographic conditions
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and the chromatograms were recorded. Also, 10 μl aliquots of the prepared
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lozenge dilutions were diluted and chromatographed under the conditions
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mentioned before and the chromatograms were recorded. Recovery percent was

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14 calculated for each dosage form using the corresponding regression equations.
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18 3. Results and discussion
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20 ATR-FTIR is a type of vibrational spectroscopy that can be used for
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22 collecting high-resolution spectral data over a wide range of wavelengths and it
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24 is the preferred infrared spectroscopic method in the laboratories. It works by
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26 quantifying how much of the infrared light is absorbed by the bonds of vibrating
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28 molecules to produce a molecular fingerprint. This is why the obtained spectra
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30 are considered as fingerprints for the under-investigation compounds (7).
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34 3.1. FTIR coupled with PLSR chemometric method
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36 3.1.1. Identification of the characteristic FTIR peaks
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38 KTP/HYS and DEX/BENZ binary drug mixtures have been successfully
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40 analyzed using the proposed method. FTIR spectra of the four compounds were
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42 recorded in the concentration ranges corresponding to that reported in their
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44 pharmaceutical dosage forms. Figures (1A and 2A) display the overlay spectra of
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46 the pure compounds (KTP / HYS and DEX/ BENZ) at concentrations of (50/ 10
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48 and 75/ 50 μg mL-1), respectively. Although the concentration of KTP is five
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50 times higher than the concentration of HYS, and the concentration of DEX is one
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52 and a half times higher than the concentration of BENZ, the recorded FTIR
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54 spectra showed many characteristic differences that can be used for the
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56 identification and quantification of each compound.
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58 The FTIR spectra show characteristic bands of the investigated drugs that
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Drug Substances books series (16) and used for the interpretation of their FTIR
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spectra. Table (2) illustrates the characteristic peak positions and the vibration
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modes of the FTIR absorption bands of all drugs in comparison to the reported
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peak positions (16). It can be easily demonstrated from Figures (1A and 2A) that
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BENZ FTIR spectrum shows the characteristic band of C=O of the ester group at

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14 a wavenumber of 1685 cm-1. Also, strong absorption bands at 1330, 1100, 950,
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16 and 600 cm-1 correspond to DEX active groups with moderate overlapping with
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18 benzocaine absorption bands. The overlay spectra of KTP and HYS show strong
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20 absorbance bands in regions such as 2900 – 2500, 1480 – 1390, 1350 – 1050, and
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22 980 cm-1 which correspond to ketoprofen, and these bands are highly overlapped
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24 with hyoscine bands. While HYS shows stronger band intensities at 3650 – 2950,
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26 1580 – 1525, 1420, and 850 – 450 cm-1 which are overlapped with KTP bands.
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28 From these observations and the infrared bands characteristics, we can conclude
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30 that the obtained spectra were representative and provide a signature of each
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32 compound.
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36 3.1.2. Optimum pre-processing steps
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38 The multivariate PLSR calibration models were constructed for the
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40 quantitative analysis of (KTP / HYS and DEX/ BENZ) in their binary mixtures
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42 and the commercial dosage forms. The PLS Modelling based on specific spectral
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44 regions and their pre-processing were done to focus on the important areas of
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46 interest and to eliminate the moderate overlapping and the unwanted instrumental
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48 artifacts. Several pre-processing calculations (such as detrending, normalization,
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50 baseline corrections, multiple scattering, and derivative algorithms) have been
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52 tried either alone or in combination to select the optimum spectral treatment. In
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54 each experiment, the value of RMSECV was computed and the pre-processing
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56 step that gives the lowest RMSECV acquired from a maximum of 7 principal
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58 components, was selected as the optimum pre-processing step for further
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calculation (17). The smallest RMSECV was obtained after using the simple
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baseline correction to remove some effects of light scattering.
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Also, the areas that may be affected by atmospheric conditions such as CO2
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gas and humidity have been cut off and ignored during the PLS model
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calculations. So that the area between 4600 – 4300 and 2600 – 1800 cm-1 has

13
14 been cut off for the mixtures of BENZ and DEX as shown in figure (2B). The
15
16 regions between 4200- 2600 and 1800- 400 cm-1 were chosen for the building of
17
18 the PLSR calculations as they were the most informative regions without
19
20 instrument artifacts or atmospheric interference. The same was conducted for
21
22 HYS and KTP mixtures, the regions between 4600 – 4200 and 2500 – 1850 cm-1
23
24 have been cut off and ignored while the regions between 4200- 2500 and 1850-
25
26 400 cm-1 were chosen for the building of the PLSR calculations as they were the
27
28 most informative regions without instrument artifacts or atmospheric interference
29
30 as shown in figure (1B).
31
32
33
34 3.1.3. PLSR Modelling
35
36 The multilevel partial factorial design consisting of (Nk) number of
37
38 samples. Where N represents the concentration levels of the investigated analytes
39
40 and k is the number of analytes. Each model consists of two analytes (k=2) with
41
42 5 concentration values of each analyte (N equals 5). Such design suggests 52 = 25
43
44 experiments to get a predictive PLSR model with 15 calibration samples and 10
45
46 validation samples distributed all over the concentration space of the analytes.
47
48 Figures (1B and 2B) show the overlay spectra of the 25 mixtures prepared for the
49
50 binary mixtures of KTP / HYS and DEX/ BENZ, respectively. The calibration
51
52 sets of uncorrelated 15 drug mixtures were selected randomly. To facilitate a good
53
54 estimation of experimental errors, the experimental design was constructed to
55
56 contain some concentration replicates.
57
58 Table (1) shows the prepared calibration concentrations that were selected
59
60 to cover the concentration range of the investigated analytes while keeping the
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4
same ratios of the binary mixtures in the market samples. It was efficiently used
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for the quantification of KTP / HYS with a linear range of 5 – 75 µg ml-1 for HYS
7
8
and 10– 100 µg ml-1 for KTP that covers the reported concentrations in their
9
10
pharmaceutical dosage form. Whereas DEX/ BENZ were analyzed in a
11
12
concentration range of 10- 100 µg ml-1 for each drug that covers the reported

13
14 concentrations in their pharmaceutical dosage form.
15
16 PLSR calculations could transform a large number of original variables to
17
18 a small number of orthogonal variables named “principal components (PCs)”.
19
20 Determination of the optimum number of principal components (factors) is the
21
22 key step in PLSR model building as the preliminary PCs are highly informative,
23
24 while the latter ones contain the noise. That is why we did not take the latter PCs
25
26 into account during building the models. The best number of principal
27
28 components selected for PLSR calibration was automatically adjusted by the
29
30 Unscrambler X software using the values of predicted residual error sum of
31
32 squares (PRESS). The PRESS value has to be as small as possible by the proper
33
34 choice of the appropriate spectral range to be used for model building (18).
35
36 Also, the value of the root-mean-square-error-of-cross-validation
37
38 (RMSECV) denotes the precision and accuracy of the model prediction.
39
40 Moreover, it can be considered as a diagnostic test for examining the errors in the
41
42 prediction set concentrations. The method developed by Haaland and Thomas
43
44 was utilized for the selection of the best number of principal components (PCS)
45
46 by using each PC to design the PLSR model then the values of RMSECV were
47
48 calculated. The PC that produces a small difference between the obtained
49
50 RMSECV and the minimum RMSECV was selected (19), so that the best
51
52 principal component value (factors) needed to build the regression model for KTP
53
54 and HYS were two. Also, the PCs needed to build the regression model for DEX
55
56 and BENZ were two as shown in figure (1S).
57
58 One of the disadvantages when using multivariate regressions is the over-
59
60 fitting of the calibration models. This means that the used data creates an
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optimistic result on the calibration set, but this model would give bad results when
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used with other data sets of similar analytes. This disadvantage can be prevented
7
8
by applying the internal validation process called the “leave-out-one cross-
9
10
validation method” (20) in which the PLSR model calculations with (1-7) factors
11
12
were performed using all the calibration samples set except one sample at a time.

13
14 This process repeated automatically by the software till each sample was left out
15
16 one time. Then, the variation between the predicted concentrations and the actual
17
18 concentrations for each left-out sample was calculated. The sum of squares of
19
20 these calculated variations is called PRESS. The smaller values of PRESS mean
21
22 a better predictive ability of the calibration model so that the over-fitting doesn’t
23
24 occur, and the model shows a high predictive ability as the obtained PRESS
25
26 values for (KTP / HYS and DEX/ BENZ) mixtures were relatively small.
27
28 Another way to ensure the predictive ability of the model was determined
29
30 by obtaining a linear relationship between the actual and the predicted variables
31
32 by plotting the reference concentrations against the corresponding predicted
33
34 concentrations as shown in Figure (2S). A satisfactory regression coefficients (r2)
35
36 (0.9998, 0.9994, 0.9855, and 0.9895) and good recovery percents (99.5, 99.89,
37
38 100.37, and 100.85) were obtained for KTP, HYS, DEX, and BENZ,
39
40 respectively, which means a high predictive power of the developed models.
41
42 Table (3) summarizes the calibration information of the chemometric models.
43
44 RMSEC indicates the distribution of the calibration concentration around the
45
46 regression line. The calculated RMSEC values and the standard error of
47
48 calibration (SEC) values were very close which means insignificant BIAS. The
49
50 calculated small values of BIAS mean a low acceptable prediction error.
51
52 The validation set was prepared using ten mixtures and it was included in
53
54 model building and its concentrations were within its range as shown in Table
55
56 (1). The validation set results showed good recovery percent in the range of 97.8
57
58 to 101 % and the relative standard deviations (RSD) were less than two,
59
60 indicating high predictive abilities of the PLSR models as shown in table (4).
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The developed models were applied to the experimental set spiked with
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excipients. Each mixture was measured three times to estimate the interference
7
8
of excipients on the predictive ability of the models. The multivariate models
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10
were applied, and good recoveries were obtained in the range of 97- 102 %,
11
12
indicating limited interference of the excipients that doesn’t affect the PLSR

13
14 model performance as shown in Table (1S). This limited interference may be due
15
16 to the filtration step that removed undissolved excipients and their very low
17
18 concentration (0.02 %) in comparison to the main active components. Also, the
19
20 excipients chemical structures that have a limited number of functional groups
21
22 that may affect the fingerprints of the under-investigation compounds.
23
24
25
26 3.2. HPLC method
27
28 Several experiments have been performed aiming for the selection of the
29
30 optimum conditions for quantification of the investigated compounds in their
31
32 binary mixtures for (KTP / HYS and DEX/ BENZ).
33
34 Concerning the first combination of KTP and HYS; The IUPAC name of
35
36 ketoprofen is 2-(3-benzoylphenyl)propanoic acid while that of hyoscine butyl
37
38 bromide is (1R,2R,4S,5S,7R)- 9 - butyl – 7 -{[(2S)-3- hydroxy-2 – phenyl
39
40 propanoyl] oxy-9 methyl -3-oxa-9 azatricyclo [3.3.1.0²,⁴] nonan-9-ium bromide,
41
42 their chemical structures indicate the presence of ionizable groups so that the
43
44 change of pH significantly affects their retention times. Mobile phases with
45
46 different pH values have been tried and the separation with a good resolution was
47
48 attained at pH of 3. At pH value of 3, the distribution coefficient (log D) of KTP
49
50 is (2.82) while log D of HYS is (-1.94). Log D is the log of distribution coefficient
51
52 at certain pH which is not a constant value as it varies according to the
53
54 proteogenic nature of the molecules. So that log D of the compounds at pH 3
55
56 gives an indication of lipophilicity (21) of KTP and HYS at the pH of the mobile
57
58 phase that shows higher lipophilicity of KTP. This difference in lipophilicity
59
60 allowed better resolution of KTP and HYS. Different mobile phase components
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have been examined at different ratios such as methanol and ethanol with
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of pH 3 but unfortunately, the peaks separation, symmetry, and tailing factors
7
8
were not satisfactory. That is why acetonitrile was used which gives a better
9
10
separation, resolution with a good tailing factor. Unfortunately, acetonitrile is not
11
12
a green solvent and the developed HPLC method was not green. The method for

13
14 separation of KTP/ HYS mixtures has been finalized using acetonitrile: 0.02 M
15
16 sodium dihydrogen phosphate buffer (50: 50 v/v) and the pH value was adjusted
17
18 to 3 using orthophosphoric acid. The flow rate was 1 mL min-1 and the
19
20 wavenumber of UV detection was 220 nm. The obtained chromatogram shows a
21
22 retention time of 6.09 ± 0.1 min using a linear concentration range of 50 -1000
23
24 μg mL-1 for KTP, and a retention time of 3.85 ± 0.1 min using a linear
25
26 concentration range of 10 -200 μg mL-1 for HYS as shown in figure (3). Tables
27
28 (2S) show the system suitability parameters for the separation of KTP/ HYS
29
30 mixtures using the proposed HPLC method. The chromatographic analysis was
31
32 carefully validated in terms of linearity, range, accuracy, repeatability, and
33
34 ruggedness and the obtained results indicate a valid method for quantification of
35
36 KTP/ HYS as shown in table (3S).
37
38 Concerning the second combination of DEX and BENZ; The IUPAC
39
40 name of dextromethorphan hydrobromide is hydrogen (1S,9S,10S)-4-methoxy-
41
42 17-methyl -17- azatetracyclo [7.5.3.0¹,¹⁰.0²,⁷] heptadeca -2,4,6-triene hydrate
43
44 bromide while that of benzocaine is ethyl 4-aminobenzoate, their structures
45
46 indicate the presence of ionizable groups so that the change of pH significantly
47
48 affect their retention times.
49
50 Different pH values have been tried and the separation with a good
51
52 resolution was attained at pH of 4. At pH value of 4, log D of DEX is (0.01) while
53
54 log D of BENZ is (1.5). So log D of the compounds at pH 4 gives an indication
55
56 of lipophilicity (21) of DEX and BENZ at the pH of the mobile phase that shows
57
58 higher lipophilicity of BENZ. Different mobile phase combinations were tried
59
60 using methanol, ethanol, and acetonitrile. From the green analytical method point
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of view, acetonitrile was the final option. Unfortunately, acetonitrile was
DOI:the best
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for the separation of DEX and BENZ. The method for separation of DEX/BENZ
7
8
mixtures has been achieved by using acetonitrile: 0.02 M sodium dihydrogen
9
10
phosphate buffer (40:60 v/v) and the pH value was adjusted to 4 using
11
12
orthophosphoric acid. The flow rate was 1 mL min-1 and the wavenumber of UV

13
14 detection was 230 nm. The obtained HPLC chromatogram shows a retention time
15
16 of 4.34 ± 0.02 min for DEX with a linear concentration range of 5 -100 μg mL-1
17
18 for DEX, and a retention time of 7.88 ± 0.04 min using a linear concentration
19
20 range of 5 -100 μg mL-1 as shown in figure (4). Tables (4S). shows the system
21
22 suitability parameters of the developed method for the separation of DEX/BENZ
23
24 mixtures. The chromatographic analysis was carefully validated in terms of
25
26 linearity, range, accuracy, repeatability, and ruggedness and the obtained results
27
28 indicate a valid method for the quantification of DEX/ BENZ as shown in Table
29
30 (5S).
31
32 The method specificity was determined by chromatographic separation of
33
34 different laboratory prepared mixtures containing KTP/ HYS or DEX/ BENZ
35
36 using the same ratios of their pharmaceutical dosage forms, then the
37
38 corresponding concentrations were calculated using the regression equations. The
39
40 obtained specificity values confirmed the applicability of separation methods as
41
42 shown in Tables (3S and 5S).
43
44 The under-investigation analytes peaks purity was assessed in all
45
46 chromatograms using the photodiode array detector. It can be used to measure
47
48 the peak homogeneity and absence of impurities. It is the weighted average of all
49
50 Spectral Contrast Angles calculated by comparing the spectra from all data points
51
52 in the integrated peak against the peak apex spectrum (22). As shown in figures
53
54 (5 and 6), the peak purity angle is smaller than the calculated purity threshold
55
56 indicating that the peaks are spectrally homogeneous and of high purity in all the
57
58 analyzed samples.
59
60
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3.3.1. FTIR coupled with PLSR method
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The calibration models were applied for the quantification of the drugs
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binary mixtures in their commercial dosage forms. Spasmofen® ampoules,
11
12
labeled to include 100 mg ketoprofen and 20 mg hyoscine butylbromide /2 mL,

13
14 were analyzed for quantification of HYS and KTP. Once the dosage form is
15
16 emptied and diluted, it can be directly placed on the surface of the ATR unit and
17
18 scanned to record their spectra in less than one minute then the proposed PLSR
19
20 models were applied. Cēpacol® Extra strength lozenges labeled to contain 7.5 mg
21
22 benzocaine and 5 mg dextromethorphan HBr / lozenge, were analyzed for
23
24 quantification of BENZ and DEX. Once the ground lozenges are dissolved in
25
26 methanol, filtered, and diluted, they can be directly placed over the surface of the
27
28 ATR unit and scanned to record their spectra in less than one minute then the
29
30 proposed PLSR models were applied.
31
32 The proposed FTIR quantitative analysis combined with the PLSR
33
34 chemometric calculations was successfully used for the quantification of the four
35
36 active pharmaceutical components in their commercial pharmaceutical dosage
37
38 forms and good recovery percent in the ranges of 98 – 100 % were obtained as
39
40 shown in Table (5). The obtained results indicate high applicability of the
41
42 chemometric method and the ability to get results in less than one minute with
43
44 high accuracy and precision.
45
46
47
48 3.3.2. HPLC method
49
50 The developed chromatographic separation methods were utilized for the
51
52 analysis of the four active pharmaceutical ingredients in their binary mixtures
53
54 with high accuracy. Spasmofen® ampoules and Cēpacol® Extra strength lozenges
55
56 were prepared as mentioned before. Once the samples were prepared, they can be
57
58 directly injected using the optimized chromatographic conditions to get the result
59
60 within 15 minutes run time. The method showed high accuracy for the
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quantification of the active components in the pharmaceutical dosageDOI:forms as
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summarized in Table (5).
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Finally, the results obtained by using the multivariate chemometric
9
10
methods and the HPLC chromatographic separations for the analysis of KTP /
11
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HYS and DEX/ BENZ were statistically compared as shown in Table (6). The

13
14 estimated t and F values did not exceed the tabulated values which mean
15
16 insignificant differences between the proposed methods’ results. It can be
17
18 concluded that the proposed ATR-FTIR chemometric analysis and the HPLC
19
20 method are equally efficient in the analysis of the four drugs and they are suitable
21
22 for the quantitative analysis of these active pharmaceutical components in their
23
24 pharmaceutical formulations as quality control tests.
25
26
27
28 3.4. Challenges and opportunities of the method in the light of GAC
29
30 principles
31
32 It can be concluded from the obtained results that ATR-FTIR coupled to
33
34 PLSR calculations has the same performance as the HPLC method in the analysis
35
36 of pharmaceutical compounds in terms of accuracy, precision, repeatability, and
37
38 applicability as shown in Table (6). Chromatographic techniques such as HPLC
39
40 is the most important and useful technique for the analysis of many analytes and
41
42 active pharmaceutical ingredients. Nevertheless, from the GAC point of view (2),
43
44 HPLC methods suffer from many difficulties such as long operation and analysis
45
46 time, large amounts of solvents used, high amount of waste generated, operator
47
48 risk, high energy consumption (more than 0.1 kWh per sample), several method
49
50 optimization trials, high cost and difficult integration with intelligent techniques
51
52 such as chemometrics. So that there are several publications and trials to mitigate
53
54 these difficulties (23).
55
56 On the other hand, vibrational spectroscopy methods such as ATR-FTIR
57
58 techniques are a good alternative that can fulfill the twelve principles of GAC as
59
60 following: -
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1) Direct analytical technique: FTIR analysis is a straightforward technique that
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can be used for direct quantification of the investigated compounds without
7
8
multiple sampling steps or extraction. Most of the active functional groups
9
10
have relative sharp absorption peaks in the mid-IR region between 1500 – 400
11
12
nm. Each compound has a unique structure permitting a unique complicated

13
14 series of absorption bands in this area which acts as a fingerprint of this
15
16 compound.
17
18 2) Minimal sample size and number: FTIR spectra are highly informative about
19
20 the structure and concentration of the compounds and coupling with intelligent
21
22 techniques such as chemometric tools permits the collection of all the available
23
24 information about the compounds using only one sample so that the number of
25
26 required samples is decreased in comparison to HPLC methods. HPLC is a
27
28 chromatographic separation technique that requires several trials for method
29
30 development and optimization of the separation conditions such as mobile
31
32 phase components, ratio, flow rate, pH, stationary phase, temperature,
33
34 wavelength), which is a tedious process that requires a high number of samples
35
36 and a large volume and these trials increases by increasing the complexity of
37
38 samples. So that several trials consuming a large number of samples are
39
40 required until the optimum calibration model is reached. While in the case of
41
42 FTIR analysis, the infrared spectrum is considered as a fingerprint of each
43
44 compound and it can be identified by a simple scan. These fingerprints may be
45
46 affected by the presence of multiple components in the sample, but
47
48 representative peaks still exist in their recorded spectrum. The coupling of
49
50 FTIR with chemometric tools such as PLSR permits resolving multicomponent
51
52 mixtures using only one sample without several trials so this technique
53
54 consumed a smaller number of samples than HPLC.
55
56 3) In situ measurements: many of the FTIR analyzers are portable permitting an
57
58 instantaneous collection of data on its site allowing the transfer from benchtop
59
60 experiments to on-site measurements in a cost-effective way.
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4) Integration of analytical processes: FTIR analysis is a fully integrative
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analytical process that can be coupled with chemometric tools permitting the
7
8
collection of the required data thus minimizing energy consumption, samples
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10
used and waste generated.
11
12
5) Automated and miniaturized methods: FTIR devices either portable or

13
14 benchtop are successfully coupled with recent advances in microelectronics,
15
16 microfabrication, and nanotechnology such as surface-enhanced infrared
17
18 spectroscopy which is considered as lab on Chip devices. HPLC devices with
19
20 autosamplers have the same advantage.
21
22 6) Avoiding derivatization: FTIR analysis is one of the few analytical
23
24 techniques that have the ability for the analysis of samples without the need for
25
26 derivatization. Each analyte has its vibrational frequencies in the mid-IR region
27
28 so that each analyte has a characteristic unique absorption pattern permitting
29
30 direct analysis without any sample derivatization.
31
32 7) Minimal analytical waste: FTIR analysis is a direct analytical technique that
33
34 can be used for the analysis of samples even in its solid form without solvents.
35
36 The combination with multivariate chemometric tools decreases the number of
37
38 required samples, decreasing the generated waste. Also, small sample volumes
39
40 are required (only 20 μL) and short operation time as the analysis time of
41
42 unknown samples is done in only one-minute leading to less solvent
43
44 consumption and waste generation.
45
46 8) Multi-analyte method: In FTIR analysis, each compound can be identified
47
48 using its characteristic peaks in the fingerprint region so that multiple analytes
49
50 can be analyzed especially that coupling with chemometric tools can solve
51
52 possible peaks overlapping and facilitate the collection of large amounts of
53
54 information from each sample. HPLC devices can analyze multiple
55
56 components with high accuracy but the method development for multiple
57
58 components is a tedious practice and consumes a very long time with larger
59
60 volumes of waste.
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analysis and rapid results permit minimal energy consumption as the FTIR
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device can perform 45 infrared scans for one sample in one minute. Portable
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FTIR devices work by Batteries, while all HPLC devices are benchtop devices,
11
12
operated for long times, and consume a high amount of energy.

13
14 10) Reagents obtained from a renewable source: Most of the solvents used in
15
16 the HPLC analysis are organic solvents which increase the organic waste.
17
18 While a wide range of solvents can be used in the FTIR analysis permitting the
19
20 ability for selection of green solvents. The developed methods for the
21
22 investigated compounds use methanol as a solvent, this is due to the limited
23
24 solubility of the compounds in a greener solvent. But many researchers
25
26 consider methanol as a green solvent because it is simple alcohol, produced
27
28 from natural sources such as biomass, coal, industrial emissions, and landfill
29
30 gas.
31
32 11) Elimination of toxic reagents: The developed method requires only 20 L
33
34 of the sample volume in each mixture analysis, this is the total solvent volume
35
36 consumed in each run. While HPLC separation requires a continuous flow of
37
38 the mobile phase for hours producing large volumes of organic solvent waste.
39
40 Also, FTIR devices have the ability for analyzing samples in all its physical
41
42 forms such as gas, solid, and liquid states. In the case of gas or solid samples,
43
44 there is no need for any solvent.
45
46 12) Operator safety: FTIR spectroscopy can be considered as one of the safest
47
48 analytical techniques with many advantages to the operator in terms of fast
49
50 analysis, multicomponent analysis in a single run, minimal sample volumes,
51
52 some methods can be operated without solvents and minimal waste. While
53
54 HPLC methods are based on the mobile phases of organic solvents. Most of
55
56 these solvents are volatile and the operator may be exposed to these vapors for
57
58 a long time. This can be solved by using face masks with filters.
59
60
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In conclusion, all the developed methods in this work achieved satisfactory
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results demonstrating that the HPLC method and ATR-FTIR combined with
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PLSR chemometric tools have the same performance for pharmaceutical analysis.
11
12
We can conclude from the above results and discussion that the utilization of

13
14 FTIR analysis coupled to chemometrics calculations is a promising analytical tool
15
16 aiming to decrease the difficulty of analyzing pharmaceuticals in full green
17
18 performance. It can help to decentralize analytical measurements with fewer
19
20 obstacles in terms of short operation times, portability, less cost, less organic
21
22 waste, and operator safety in comparison to the traditional methodologies like
23
24 HPLC. In spite of the great attempts aiming to make the mentioned analytical
25
26 chromatographic separations as green as possible by means of reducing or
27
28 replacing the organic waste generated. Practically, the quality control processes
29
30 of pharmaceutical production by HPLC is a tedious analytical practice, mostly in
31
32 the separation step, as several overlapping parameters must be optimized through
33
34 multiple chromatographic runs during the method development to achieve a
35
36 successful separation. On the other hand, vibrational spectroscopic techniques
37
38 especially ATR-FTIR offer the highest level of integration with the green
39
40 analytical chemistry principles and also agree well with the protocol of “Process
41
42 Analytical Technology” in terms of the ease of manipulation, limited waste,
43
44 operator’s safety, analysis of samples as it is, portability and structural related
45
46 analysis (fingerprints).
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bambuterol and terbutaline in human urine samples using ATR-FTIR coupled
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53 11. Eid SM, Abd El-Rahman MK, Elghobashy MR, Kelani KM. Attenuated
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5 14. Butler HJ, Smith BR, Fritzsch R, Radhakrishnan P, Palmer DS, Baker MJ.
6 Optimised spectral pre-processing for discrimination of biofluids via ATR-FTIR
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spectroscopy. The Analyst. 2018;143(24):6121-34.
9 15. Branch SK. Guidelines from the International Conference on
10 Harmonisation (ICH). Journal of Pharmaceutical and Biomedical Analysis.
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2005;38(5):798-805.
16. Analytical Profiles of Drug Substances1990.

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14 17. Rinnan Å. Pre-processing in vibrational spectroscopy – when, why and
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16 how. Anal Methods. 2014;6(18):7124-9.
17 18. Theophile T. Infrared Spectroscopy - Life and Biomedical Sciences2012.
18 19. Geladi P, Kowalski BR. Partial least-squares regression: a tutorial.
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20 Analytica Chimica Acta. 1986;185:1-17.
21 20. Brereton RG. Chemometrics: Data Analysis for the Laboratory and
22 Chemical Plant: Wiley; 2003.
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24 21. Kah M, Brown CD. LogD: Lipophilicity for ionisable compounds.
25 Chemosphere. 2008;72(10):1401-8.
26 22. Papadoyannis IN, Gika HG. Peak Purity Determination with a Diode Array
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28 Detector. Journal of Liquid Chromatography & Related Technologies.
29 2007;27(6):1083-92.
30 23. Abd El-Rahman MK, Zaazaa HE, ElDin NB, Moustafa AA. Just-Dip-It
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32 (Potentiometric Ion-Selective Electrode): An Innovative Way of Greening
33 Analytical Chemistry. ACS Sustainable Chemistry & Engineering.
34 2016;4(6):3122-32.
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List of tables View Article Online
DOI: 10.1039/D0AY01749C
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6 Table (1): the calibration and validation sets designs and concentration ratios of
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the binary mixtures of KTP/ HYS and BENZ/ DEX
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Mixture Concentrations (μg ml-1) Mixture Concentrations (μg ml-1)
12 Number* KTP HYS Number* DEX BENZ

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14 1 50 25 1 50 50
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16 2 50 75 2 75 100
17 3 10 75 3 100 10
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19 4 10 5 4 10 10
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21 5 100 50 5 10 50
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23 6 25 5 6 50 75
24 7 100 25 7 75 10
25
26 8 50 50 8 10 75
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28 9 25 50 9 75 25
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30 10 25 10 10 25 25
31 11 75 5 11 25 50
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33 12 100 10 12 50 10
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35 13 75 25 13 10 25
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37 14 50 5 14 25 10
38 15 100 5 15 10 100
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40 16 100 75 16 100 100
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42 17 10 10 17 100 50
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44 18 75 75 18 50 25
45 19 10 25 19 25 100
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47 20 50 10 20 100 25
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49 21 75 10 21 25 75
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51 22 75 50 22 75 75
52 23 25 75 23 75 50
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54 24 10 50 24 50 100
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56 25 25 25 25 100 75
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58 * The highlighted mixtures with blue colors represent the 15 mixtures of the
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calibration set while the remaining represent the validation set mixtures.
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Table (2): Infrared absorption bands and different modes of vibrations for
View Article Online
DOI: 10.1039/D0AY01749C
5 benzocaine, dextromethorphan HBr, ketoprofen and hyoscine butyl bromide.

6
7 Reported and/or Present
Studied
8 bands assignments expected values method values
9 drug
10 Peak (cm-1) Peak (cm-1)
11 NH2 asymmetric stretch 3423 3454
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CH asymmetric stretch 3000 2989

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C=O stretching 1684 1685
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16 NH2 bending 1636 1610
Benzocaine
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18 Ring asymmetric stretch 1544 1546
19 C-N Stretching 1312 1313
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21 C-O stretching 1281 1280
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CH asymmetric stretch 1120 1115
24 CCC symmetric stretching 846 847
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26 CCC asymmetric stretching 773 772
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Aromatic C-H stretching 3000-3100 3000
Dextromethorphan HBr
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30 C=C stretching 1608 1608
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33 CH3-O- Stretching 2921 2924
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N-CH stretching 2780 2775
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38 C-N stretching 1445 1420
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40 O-H stretching 3600 - 3200 3300
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42 C-H stretching of aromatic 3020 masked
43 C-H stretching of CH3 group 2970, 2930 2970, 2920
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45 C=O stretching of acid 1695 1680
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C=O stretching of ketone 1655 1650
Ketoprofen
48 C=C stretching of aromatic 1595, 1580, 1599, 1535,

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50 ring 1455 1470
51 C-H deformation of CH3
52 1440 1435
53 (asymmetric)
54 C-H deformation of CH3
55 1370 1377
56 (symmetric)
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C-H deformation of aromatic
860 - 690 859 - 696
59 rings (several bands)
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OH stretching 3350 3360
View Article Online
4 DOI: 10.1039/D0AY01749C
hyoscine butyl bromide CH Stretching 2950 2950

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7 N-CH3 2810 2814
8 C=O (in ester) 1728 1725
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10 C=C (aromatic) 1600 1599
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C-O-C (ether) 1166, 1045 1164, 1048

13 5H (Monosubstituted
14 780, 735, 700 779, 734, 650
aromatic
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Table (3): The calibration data and PLSR model validation parameters
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27 Chemometric PLSR model
Parameters
28 BENZ DEX HYS KTP
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30 Number of samples 15 15 15 15
31 Range (µg/ml) 10– 100 10– 100 5– 75 10– 100
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33 Slope 0.9837 0.9803 1.0030 0.9972
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35
Offset (Intercept) 0.9303 0.7856 0.1738 0.0893
36 Correlation 0.9947 0.9927 0.9996 0.9998
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38 R2(Pearson) 0.9895 0.9855 0.9994 0.9998
39 RMSEC 6.1930 5.3336 3.3897 2.9962
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41 SEC 6.4952 5.5939 3.6687 3.1606
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43
Bias 0 0 0.00001 0.00001
44 RMSECV 4.9759 3.3368 7.7165 8.7573
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46 SECV 4.6478 3.0865 7.6984 9.0872
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Table (4): The predictive ability of the PLSR model by application to View
theArticle Online
DOI: 10.1039/D0AY01749C
5 validation set mixtures (mean recoveries (R%) ± relative standard deviation

6
7 (R.S.D))
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10 Analytes (R% ± R.S.D)* Analytes (R% ± R.S.D)*
Mixture
Mixture
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no #
no #
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BENZ DEX HYS KTP

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15 4 99.72 ± 0.71 99.02 ± 0.89 1 98.22 ± 0.11 99.25 ± 0.99

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17 6 98.80 ± 0.14 101.60 ± 0.62 2 97.58 ± 0.02 100.68 ± 0.87
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10 97.83 ± 1.44 97.89 ± 1.28 9 99.76 ± 0.23 100.83 ± 0.18
20 12 100.03 ± 0.89 98.99 ± 0.88 16 100.69 ± 0.17 101.01 ± 0.77
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22 15 98.61 ± 0.92 98.32 ± 1.96 17 98.22 ± 0.98 98.81 ± 0.96
23 17 100.33 ± 0.59 98.89 ± 0.61 19 97.89 ± 1.21 99.19 ± 1.01
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25 18 100.27 ± 1.06 99.99 ± 1.42 22 100.74 ± 0.92 99.81 ± 1.92
26 21 100.85 ± 0.84 100.44 ± 0.92 23 101.01 ± 0.67 98.47 ± 1.22
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28 24 99.70 ± 0.78 101.02 ± 0.78 24 99.89 ± 0.33 99.22 ± 0.13
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25 101.01 ± 0.55 99.22 ± 0.93 25 99.33 ± 1.22 97.52 ± 1.69
31 # The concentration levels provided in (Table 1).
32
33 * The results are average of three replicates.
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40 Table (5): Application of PLSR model for quantification of benzocaine,
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42 dextromethorphan HBr, ketoprofen and hyoscine butyl bromide in their
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pharmaceutical dosage form.
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% Recovery (in reference to labeled amount) ± S.D *
48 Method of Cēpacol® Extra strength
49 Spasmofen® ampoules
50 analysis lozenges
51 BENZ DEX HYS KTP
52
53 PLSR Method 99.67 ± 1.66 98.67 ± 1.89 98.89 ± 1.32 98.99 ± 0.97
54
55 HPLC Method 99.74 ± 1.56 99.86 ± 0.43 101.72 ± 0.60 101.73 ± 1.07
56
57 * The results are average of three determinations
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Table (6): Comparison of the proposed methods for determination of benzocaine,View Article Online
DOI: 10.1039/D0AY01749C
5 dextromethorphan HBr, ketoprofen and hyoscine butyl bromide with the

6
7 developed HPLC method.
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10 FTIR coupled with PLSR
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Developed HPLC method
Parameter Chemometric method
12
BENZ DEX HYS KTP BENZ DEX HYS KTP

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14
M.R % 100.85 100.37 99.89 99.96 100.61 100.73 99.63 99.50
15
16 S. D 1.17 1.39 0.26 1.18 1.61 2.03 0.92 1.40
17
18 N 15 15 15 15 6 6 5 6
19 Standard
20 0.302 0.359 0.067 0.305 0.657 0.829 0.041 0.572
21 error
22 Student’s t 0.382 0.471 1.026 0.767
23
24 test b (2.09) (2.09) (2.09) (2.09)
25
1.894 2.133 0.080 0.710
26 F test b
27 (4.68) (2.96) (2.96) (2.96)
28
29
30 b The numbers in parenthesis are the tabulated values of F and t at the 95%
31
32 confidence level.
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List of Figures View Article Online
DOI: 10.1039/D0AY01749C
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8 (A)
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Cut off
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Cut off
region
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43 Figure (1): (A) The FTIR spectra of 10 μg mL-1 hyoscine and 50 μg mL-1
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45 ketoprofen at wavenumber 4600- 400 cm-1 and the chemical structures of
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47
compounds. (B) Overlay spectra of the 25 mixtures of hyoscine and ketoprofen.
48 The highlighted area is the cut off regions that we did not use during building of
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the PLSR model.
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4 DOI: 10.1039/D0AY01749C
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Cut off
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42 Figure (2): (A) The FTIR spectra of 50 μg mL-1 benzocaine and 75 μg mL-1
43 dextromethorphan HBr at wavenumber 4600- 400 cm-1 and the chemical
44
45 structures of compounds. (B) Overlay spectra of the 25 mixtures of benzocaine
46 and dextromethorphan HBr. The highlighted area is the cut off regions that we
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48 did not use during building of the PLSR model.
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28 Figure (3): HPLC chromatogram of HYS (10 μg mL-1) at (RT= 3.356 min) and
29 KTP (50 μg mL-1) at (RT= 5.530 min) in Spasmofen ampoules.
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56 Figure (4): HPLC chromatogram of DEX (50 μg mL-1) at (RT= 4.360 min) and
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58 BENZ (75 μg mL-1) at (RT= 7.841min) in Cēpacol® Extra strength lozenges.
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46 Figure (5): 3D plot of the chromatogram of separation of HYS (10 μg mL-1) at
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48 (RT= 3.356 min) and KTP (50 μg mL-1) at (RT= 5.530 min), and their purity plots
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showing the purity angle and purity threshold.
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47 Figure (6): 3D plot of the chromatogram of separation of DEX (50 μg mL-1) at
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49 (RT= 4.360 min) and BENZ (75 μg mL-1) at (RT= 7.841min), and their purity
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51 plots showing the purity angle and purity threshold.
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5 benzocaine, dextromethorphan HBr, ketoprofen and hyoscine butyl bromide.

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48 C=C stretching of aromatic 1595, 1580, 1599, 1535,

hyoscine butyl bromide CH Stretching 2950 2950

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5 validation set mixtures (mean recoveries (R%) ± relative standard deviation

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15 4 99.72 ± 0.71 99.02 ± 0.89 1 98.22 ± 0.11 99.25 ± 0.99

5 dextromethorphan HBr, ketoprofen and hyoscine butyl bromide with the

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