Numerous researches have been carried out to determine the biological effects of the raspberry components, thus obtaining numerous results. For example, the ellagic acid is reported to inhibit tumor inductions in the liver, lungs and esophagus only in tumour cell lines and animal models [10Ravai M. Quality characteristics of raspberries and blackberries. Cereal Foods World 1996; 41: 773-5.]. It is presumed that RK might influence the lipid metabolism in ways similar to capsaicin and synephrine. The effects of RK on obesity and lipid metabolism were examined by Morimoto et al. [11Morimoto C, Satoh Y, Hara M, Inoue S, Tsujita T, Okuda H. Anti-obese action of raspberry ketone. Life Sci 2005; 77(2): 194-204.
[http://dx.doi.org/10.1016/j.lfs.2004.12.029] [PMID: 15862604] ].

RK is found naturally in many foods, most notably raspberries, but also in Rheum officinale [12Tateiwa JI, Horiuchi H, Hashimoto K, Yamauchi T, Uemura S. Cation-exchanged montmorillonite-catalyzed facile Friedel-Crafts alkylation of hydroxy and methoxy aromatics with 4-hydroxybutan-2-one to produce raspberry ketone and some pharmaceutically active compounds. J Org Chem 1994; 59: 5901-4.
[http://dx.doi.org/10.1021/jo00099a017] ].

Dietary intake is estimated to be about 0.42mg/kg bodyweight [13Park KS. Raspberry ketone increases both lipolysis and fatty acid oxidation in 3T3-L1 adipocytes. Planta Med 2010; 76(15): 1654-8.
[http://dx.doi.org/10.1055/s-0030-1249860] [PMID: 20425690] ], consumed through processed foods where raspberry ketone is added as a flavoring agent.

Raspberry ketone quantitative determination has been performed using using polarographic technique polarography [14Zhang ZH, Xu LM, Yao BQ. Spectrophotometry determination of raspberry ketone. J Beijing Inst Petrochemieal Tech 2005; 13: 15-6.], spectrometry [15Wang L, Wang Y, Zhang Y, Meng X, Yang L, Zhang F. Determination of raspberry ketone in raspberry by high performance liquid chromatography tandem mass spectrometry. International Conference on Human Health and Biomedical Engineering August 19-22, 2011; Jilin, China. ] and liquid chromatography - mass spectrometry [16Al-Othman ZA, Al-Warthan A, Aboul-Enein HY, Al Za’abi M, Ali I. Mechanistic approaches of PhE and PPF columns separation for rasberry ketone and caffeine. J Liq Chromatogr Relat Technol 2015; 38: 1324-32.
[http://dx.doi.org/10.1080/10826076.2015.1048874] -18Aprea E, Biasioli F, Gasperi F. Volatile compounds of raspberry fruit: from analytical methods to biological role and sensory impact. Molecules 2015; 20(2): 2445-74.
[http://dx.doi.org/10.3390/molecules20022445] [PMID: 25647579] ]. The-HPLC separation of raspberry ketone and caffeine on phenyl columns (PhE and PPF) has also been described [16Al-Othman ZA, Al-Warthan A, Aboul-Enein HY, Al Za’abi M, Ali I. Mechanistic approaches of PhE and PPF columns separation for rasberry ketone and caffeine. J Liq Chromatogr Relat Technol 2015; 38: 1324-32.
[http://dx.doi.org/10.1080/10826076.2015.1048874] ].

Caffeine (II, CAF) (3, 7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione or 1,3,7- trimethylxanthine) is a central nervous system and metabolic stimulant, which is used both recreationally and medically to reduce physical fatigue and to restore alertness when drowsiness occurs [19Nehlig A. Are we dependent upon coffee and caffeine? A review on human and animal data. Neurosci Biobehav Rev 1999; 23(4): 563-76.
[http://dx.doi.org/10.1016/S0149-7634(98)00050-5] [PMID: 10073894] -21Nkondjock A. Coffee consumption and the risk of cancer: an overview. Cancer Lett 2009; 277(2): 121-5.
[http://dx.doi.org/10.1016/j.canlet.2008.08.022] [PMID: 18834663] ]. It produces increased wakefulness, faster and clearer flow of thought, increased focus, and better general body coordination. Caffeine's most notable pharmacological effect is as a central nervous system stimulant, increasing alertness and producing agitation. It also acts as a urinary stimulant.

Caffeine is used in some headache and migraine medications, in certain dietary supplements used for weight loss and in many popular energy drinks [22Santos JR, Rangel AOSS. Determination of caffeine in coffee using low-pressure chromatography.Coffee in Health and Disease Prevention 2015; 983-91.
[http://dx.doi.org/10.1016/B978-0-12-409517-5.00108-X] ].

Caffeine quantitative determination has been performed using different techniques ranging from liquid chromatography to spectrometry [23van Koert RR, Bauer PR, Schuitema I, Sander JW, Visser GH. Caffeine and seizures: A systematic review and quantitative analysis. Epilepsy Behav 2018; 80: 37-47.
[http://dx.doi.org/10.1016/j.yebeh.2017.11.003] [PMID: 29414557] -28Corciova A, Ivanescu B. Determination of caffeine content from different pharmaceutical and natural products. Eur Chem Bull 2014; 5: 138-41.].

The main objective of this work was to develop a new procedure for the fast and accurate simultaneously determination of RK and CAF in commercial nutraceutical formulations containing 100 mg RK and 50 mg CAF per tablet, by using the Beer-Lambert law thus reducing the steps required for sample pre-treatment and providing direct FTIR measurement.

Data acquisition was performed using a Frontier Two System FT-IR spectrometer equipped with Spectrum for Windows v.5.01 (Perkin Elmer Co., Beaconsfield, Bucks, UK). The commercial softwares used were QUANT+ expert v/4.51 and Spectrum Beers’s law v.2.01 (Perkin Elmer Co.,UK) to generate data for the principal component analysis. All the FT-IR spectra were scanned between 4000 and 400 cm^-1, by averaging 32 scans for each spectrum with a resolution of 4 cm^-1 (data point resolution/interval 1 cm^-1) and were recorded with a DTGS detector.

The standards of RK, CAF and Microcrystalline Cellulose (MCC) were supplied by Sigma Aldrich. For fused KBr disk preparation a potassium bromide IR spectral grade was used (Sigma Aldrich, Steinhein, Germany). The nutraceutical formulation, Ultimate containing 100mg RK/tablet and respectively 50mg CAF/tablet, manufactured by WELLNX – Willington Delaware, USA, was purchased from the local market.

The principal excipient used in the tablet formulation is Microcrystalline Cellulose (MCC).

The standards were prepared by compressing 2.0 mg of substance with spectral grade KBr (up to 200mg), while the background was spectral grade KBr. Each spectrum was collected three times for the same cup after rotation with 120⁰. The mean of the spectra was then used in the following analysis steps.

For the calibration curve of RK, we used samples prepared by compressing a standard substance RK in spectral grade KBr, while the background was 0.5 mg MCC in KBr (calibration was made using four levels: 0.1, 0.2, 0.3 and 0.4 mg respectively). For the calibration curve of CAF, spectra were recorded from samples prepared by compressing a standard substance CAF in spectral grade KBr, while the background was 0.5 mg MCC in KBr (calibration was made using four points 0.05, 0.1, 0.15 and 0.2 mg respectively).

For the drug determination, the spectra were performed in two different ways:

• Conventional fused KBr disk spectra were recorded from the samples prepared by compressing 2.0mg of drug sample with spectral grade KBr, while the background was spectral grade KBr [Drug -KBr].
• Conventional fused KBr disk spectra were recorded from samples prepared by compressing 2.0 mg of drug sample with spectral grade KBr, while the background was 0.5mg MCC in KBr [Drug-MCC].

Experimental parameters, such as calibration methods, were compared and recommendations on the best options for RK and CAF analysis were made.