Accepted Manuscript Alternative method for chromium determination in pharmaceutical drugs by HR-CS GF AAS and direct analysis of solid samples
Eliana G. Barrera, Débora Bazanella, Paula W. Castro, Wiliam Boschetti, Maria G.R. Vale, Morgana B. Dessuy PII: DOI: Reference:
S0026-265X(16)30647-6 doi: 10.1016/j.microc.2017.02.020 MICROC 2706
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
21 November 2016 15 February 2017 16 February 2017
Please cite this article as: Eliana G. Barrera, Débora Bazanella, Paula W. Castro, Wiliam Boschetti, Maria G.R. Vale, Morgana B. Dessuy , Alternative method for chromium determination in pharmaceutical drugs by HR-CS GF AAS and direct analysis of solid samples. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Microc(2017), doi: 10.1016/j.microc.2017.02.020
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ACCEPTED MANUSCRIPT Alternative method for chromium determination in pharmaceutical drugs by HR-CS GF AAS and direct analysis of solid samples
Eliana G. Barrera, Débora Bazanella, Paula W. Castro, Wiliam Boschetti, Maria
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G. R. Vale, Morgana B. Dessuy
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ABSTRACT
High-resolution continuum source graphite furnace atomic absorption
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spectrometry (HR-CS GF AAS) was used to develop an analytical method for chromium determination in pharmaceutical drugs through direct analysis of solid
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samples. The limit of detection (LOD) and quantification (LOQ) were, respectively, 2.9 μg kg-1 and 9.7 μg kg-1 and the characteristic mass was 2.0 pg.
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Eighteen pharmaceutical drugs and three excipients were analyzed. The
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encapsulated samples were opened and only its powder content was analyzed; the tablet samples were grinded before the analysis. The results obtained from
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the direct analysis of the solid samples were in agreement with those found after total digestion of samples using a closed-vessel microwave oven device,
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with a confidence level of 95%. The Cr concentration obtained for the different capsules and tablets investigated varied between 0.05 and 1.92 mg kg -1. The proposed method is fast, reliable and simple for routine pharmaceutical analysis.
Keywords: pharmaceutical drugs, direct analysis, HR-CS GF AAS, chromium.
ACCEPTED MANUSCRIPT 1. Introduction
In the pharmaceutical industries, a variety of metals and non metals are used in the manufacture of drugs, or even used as the active pharmaceutical ingredient (API) in medicine products [1]. Thus, it is common to find metallic
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impurities on the final products originated from different sources, as raw
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materials (plants, animal proteins, etc), metal catalysts or metal reagents used
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during synthesis, excipients (stabilizers, fillers, release agents, flavors, color), manufacturing equipment and piping, bulk packaging, the environment, cleaning
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solvents, etc [2]. Considering the elements generally found in medicine formulations, chromium can be associated with potential health risks as
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chromosomal aberration, mutations and carcinogenicity, just to mention a few [3].
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Taking into account the increasing consumption of pharmaceutical drugs
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by humans, it is important to highlight that the quality control of these medicines is vital to guarantee the final consumers safety [1,4-6]. The permissible levels of
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potentially toxic elements in pharmaceuticals are usually defined by the regulatory agencies. For example, the permissible limit of Chromium impurities
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in drugs established from Brazilian Pharmacopeia [7] via oral use is 25 µg g-1, while the United States (USP) [8] and European Pharmacopeia (EP) [9] have settled a value of 1100 µg g-1, considering maximum daily dose of 10 g of drug products. Brazilian Pharmacopeia [7] indicates two methods for elements determination: the solid particles formation and the atomic spectrometry, which are also indicated by the USP [8] and the EP [9]. In this context, the atomic spectrometry techniques play an important role in order to determine the
ACCEPTED MANUSCRIPT potentially toxic elements concentration, as the solid particles formation test provides only semi-quantitative results. Therefore, analytical methods that provide trustworthy results through a fast and simple way are essential. Atomic absorption spectrometry (AAS) technique is recommended by Brazilian Pharmacopeia for As, Cd, Cr, Cu Hg, Ir,
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Mn, Ni, Os, Pb, Pd, Pt, Rh, Ru and V determination in pharmaceutical drugs [7].
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Among AAS techniques, graphite furnace (GF AAS) shows higher sensitivity
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when compared to flame AAS [10,11]. Inductively coupled plasma-optical emission spectroscopy (ICP OES) [2,12], inductively coupled plasma-mass
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spectrometry (ICP-MS) [13,14], electrothermal vaporization inductively coupled plasma optical emission spectrometry (ETV-ICP OES) [15] and laser ablation
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inductively coupled plasma-mass spectrometry (LA-ICP-MS) [16] also can be used in the analysis of pharmaceutical compounds. However, most of these
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techniques require sample pretreatments which, besides of being time
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consumable, can lead to sample losses or contamination and sensitivity decrease, mainly due to the use of different reagents and sample dilutions [10-
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15,17].
In this context, direct analysis of solid samples (SS) by graphite furnace
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atomic absorption spectrometry (SS-GF AAS) is an effective tool to overcome the issues found on the sample pretreatments [18,19]. Thus, the benefits and interests of SS-GF AAS on commercial capsules and tablets analysis arise from its simple manipulation and the possibility to be employed on industrial routine protocol. More recently, with the advent of the high-resolution continuum source AAS (HR-CS AAS), the direct analysis of solid samples by graphite furnace (HR-CS SS-GF AAS) became more consolidated and feasible [20-22].
ACCEPTED MANUSCRIPT The use of the SS-GF AAS for Cr determination is described by several authors which analyzed different matrices as medicinal plants [23], crude oil [24] tannin samples [25], biomass and its ashes [26], sunscreen samples [27], soil samples [28] and high purity polyimide samples [29]. It is noteworthy that these authors do not report the use of chemical modifiers for Cr determination. On the
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other hand, but still considering the employment of the SS-GF AAS, there are
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works that describe the use of chemical modifiers, mainly to overcome matrix
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interferences over the Cr analytical signal. The most common chemical modifier employed for Cr determination in different matrices, as soil certified reference
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materials [30], fertilizer samples [31] and vegetable oil and biodiesel samples [32], is a Mg(NO3)2 solution.
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All these works report that the direct analysis of solid sample is an efficient analytical tool, due to the elimination of many steps involved in the
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sample preparation reducing the time of analysis and the risk of contamination
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and it, also, avoids the sample dilution, increasing the sensitivity of the method. Moreover, the combination of direct analysis of solid samples with HR-CS AAS
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technique provides much more information about the spectral environmental around the analytical line, which facilitates method development and the spectral interferences and its correction. Besides these
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detection of
advantages, this technique has not been explored yet in order to perform the analysis of commercial pharmaceutical drugs capsules and tablets. The main goal of this work has been the development of a simple, fast and reliable analytical method for chromium determination in pharmaceutical drugs commercialized as capsules and/or tablets, as well as on excipients by HR-CS GF AAS using direct analysis of solid samples.
ACCEPTED MANUSCRIPT 2. Experimental
2.1. Instrumentation
All measurements were performed in a high-resolution continuum source
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graphite furnace atomic absorption spectrometer (HR-CS GF AAS), contrAA
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700 model (Analytik Jena, Jena, Germany). This instrument is equipped with a
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xenon short-arc lamp with a nominal power of 300 W operating in a hot-spot mode, a high-resolution double monochromator and a linear charge coupled
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device (CCD) array detector with 588 pixels. The analytical line at 357.868 nm was used for the measurements and the integrated absorbance (Aint) of center
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pixel (CP) and the two adjacent pixels, i.e. CP ±1, was summed and used for signal evaluation. Argon with purity of 99.996% (White Martins, São Paulo,
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Brazil) was used as purge gas. The argon flow rate was kept in 2.0 L min-1
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during all stages, except in the atomization, when it was stopped. The optimized graphite furnace temperature program for Cr determination is shown in Table 1.
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Transversely heated graphite tubes without a dosing hole (Analytik Jena, Part No. 407-152.316) and solid sampling graphite platform (Analytik Jena, Part
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No. 407–152.023), both coated with pyrolytic graphite were used in all experiments. An M2P microbalance (Sartorius, Göttingen, Germany - accuracy 0.001 mg) was used for weighing the pulverized samples directly onto the SS platforms. A pre-adjusted pair of tweezers, which is part of the SSA 6 manual solid sampling accessory (Analytik Jena, Jena, Germany), was used to transfer the SS platforms to the atomizer. The aqueous standards and digested
ACCEPTED MANUSCRIPT solutions were injected manually onto the platform using a micropipette. It was carried out 5 measurements of each sample and standard solution. A microwave reaction system, Multiwave PRO, (Anton Paar, Graz, Austria) was used for the acid digestion of the samples in order to verify the
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accuracy of the developed method.
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2.2. Reagent and solutions
Analytical grade reagents were used exclusively. Ultra-pure water with a
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specific resistivity of 18 MΩ cm−1 from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used in the preparation of standards and
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digested solutions. The nitric acid (Merck, Darmstadt, Germany) used for the preparation of standards and digestions was purified by sub-boiling distillation in
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a quartz apparatus (Kürner Analysentechnik, Rosenheim, Germany). Aqueous
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standard solutions were prepared by appropriate dilutions of a stock solution of
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1000 mg L−1 of Cr (Specsol, São Paulo, Brazil) with 0.014 mol L−1 nitric acid.
2.3. Sample preparation procedures
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2.3.1. Solid sample pretreatment
Eighteen pharmaceutical drugs and three excipients were obtained from local drugstores in Porto Alegre (Rio Grande do Sul, Brazil). The encapsulated samples were opened and its powder content was analyzed without any treatment. The tablet samples (PD11, PD12, PD13 and PD17) containing an external coating layer were grinded using an agate mortar and sieved through a
ACCEPTED MANUSCRIPT 180 µm mesh. Sample masses between 0.2 and 7.5 mg of the pharmaceutical drugs or excipients were weighted onto the SS platforms and introduced into the graphite furnace for measurement. As it is not possible to weight always the same sample mass, they were transmitted to the instrument's computer to calculate the normalized Aint (calculated for 0.5 mg of sample) after each
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measurement.
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2.3.2. Samples digestion and recovery tests
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Four samples were submitted to an acid digestion procedure in order to verify the trueness of the developed method, using a microwave system
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equipped with PTFE-TFM closed vessels of 50 mL. Approximately, 0.15 g of each sample was transferred to the vessels with further addition of 3 mL of
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concentrated nitric acid and 1 mL of concentrated hydrochloric acid. The
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samples were submitted to the following heating program, performed in two steps (temperature in ºC / ramp in min / hold in min): (i) 130 / 30 / 5; (ii) 180 / 20
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/ 5. After cooling, the vessels content were transferred to 20 mL volumetric flasks and the final volume was completed with ultrapure water. Recovery tests
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were carried out using these four digested samples. In this case, adequate aliquots of chromium standard solution were added to the samples before they were submitted to the digestion procedure.
(insert Table 1)
ACCEPTED MANUSCRIPT 3. Results and discussion 3.1. Temperature program
As chromium is known as thermally stable element, the method optimization and
the analyses of pharmaceutical samples were carried out without a
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chemical modifier. The heating program was optimized using 10 µL of an
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aqueous standard solution of Cr 10 µg L-1, which corresponds to 100 pg of
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analyte, and a commercial sample of pharmaceutical drug (PD1). Initially, pyrolysis temperatures (Tpyr) between 300 ºC and 2000 ºC were evaluated
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maintaining the atomization temperature at 2600 ºC. Figure 1 shows that the Cr Aint values for the aqueous standard solution and for the investigated sample
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are stable up to 1500 ºC and for higher temperatures the Aint values decreases due to Cr losses. In other words, it indicates that standard solution and sample
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presents similar thermal behavior. In order to guarantee the complete matrix
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elimination and to preserve the graphite furnace life time, the temperature of 1200 ºC was chosen and it was used in all further experiments. Moreover at
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1200 °C a better precision, which can be observed as the relative standard
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deviation (RSD), was obtained for the samples, RSD = 4.2 %.
(insert Figure 1)
Considering that Cr is known as a non volatile element [10], atomization temperatures (Tatom) between 2300 and 2600 °C were evaluated. It is possible to observe in Figure 2 that Cr Aint values for the PD1 sample presented a slightly increase with the temperature rising. On the other hand, the A int values
ACCEPTED MANUSCRIPT for Cr standard solution remained stable up to 2500 °C, with a small decrease at 2600 °C, which may be attributed to the Cr peak shape (Figure 3a), as at 2500 °C the analytical signal was broader and more tailing than at 2600 °C, i.e. the peak area was higher at 2500 °C than at 2600 °C. However, as a more symmetrical peak was obtained, at 2600°C, this temperature was preferable
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since a shorter integration time can be used, which can contribute to a improved
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signal to noise ratio and, consequently, lower LOD and LOQ values.
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Considering Cr atomization from sample, a higher atomization temperature is more convenient also, since it results in a higher amount of analyte atomized
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(Figure 3b).
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(insert Figure 2)
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(insert Figure 3)
Figure 4 shows two time-resolved absorbance spectra obtained using the
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optimized temperatures of 1200 and 2600 °C evaluating two different samples (PD7 and PD17). No spectral interferences and symmetric peaks are observed. Therefore, the Tpyr and Tatom used in all further measurements were 1200 and 2600 °C, respectively. It is important to mention that under these conditions the graphite furnace lifetime was approximately 330 cycles.
Furthermore,
considering the absence of spectral interferences during the samples analyses
ACCEPTED MANUSCRIPT it is clear that the pharmaceutical drugs can be directly analyzed even by a line source spectrometer.
3.2. Study of influence of sample mass
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The direct analysis of solid samples can find some difficulties related to
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inhomogeneity of samples, errors due to the use of different masses, limited
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sample size and high RSD values [33-35]. Hence, to check the influence of the sample mass introduced onto the graphite furnace under optimized conditions,
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the correlation of different masses of a pharmaceutical drug (PD1) sample and their respectively Cr Aint values were evaluated. Samples masses between 0.20
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and 7.5 mg were weighted and an adequate correlation coefficient (0.9850) was obtained. Higher amounts of samples were not investigated due to the platform
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capacity, which was reasonably full when sample masses above 7.5 mg were
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evaluated. This result indicates that the sample mass does not influence the
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analytical response, i.e., the Cr Aint.
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3.3. Figures of merit
Calibration curves were established using a blank and calibration solutions in the concentration range of 2.5 – 40 µg L-1 (25 – 400 pg). Blank measurements were carried out according to the “zero mass response” principle [18] using the empty SS platform.
ACCEPTED MANUSCRIPT The limits of detection (LOD) and quantification (LOQ), defined as three and ten times the standard deviation of ten measurements of the blank, respectively, both divided by the slope of the calibration curve. The characteristic mass (m0) is defined as the mass of the analyte corresponding to an integrated absorbance of 0.0044 s. The relative LOD and LOQ were
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calculated for 7.5 mg of sample, which corresponds to the maximum sample
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mass that can be analyzed.
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The analytical figures of merit obtained for Cr are shown in Table 2. Wollein et al. [36] measured Cr in active pharmaceutical substances with GF
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AAS using the Cr analytical line at 357.9 nm achieving a LOQ of 0.05 µg g-1 for 400 mg of digested samples. Using the same wavelength, De Paula et al. [37]
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determined Cr in pharmaceutical formulations via GF AAS using a ultrasoundassisted extraction method and obtained a LOQ of 200 ng g-1, considering a
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sample mass of 25 mg. Virgilio et al. [23], evaluating medicinal plants via HR-
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CS SS-GF AAS, determined the figures of merit for Cr using aqueous standard solutions and obtained LOQ of 11 ng g-1 and characteristic mass (m0) of 4.8 pg.
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Zmozinski et al. [25] determined Cr in tannin samples using HR-CS SS-GF AAS and obtained a m0 of 2.2 pg and LOQ of 57 ng g-1, calculated for 0.25 mg of
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sample. In other words, the figures of merit obtained in this work (m0 of 2.9 pg and LOQ of 0.39 ng g-1, calculated for a sample mass of 7.5 mg) are in agreement or even better with those reported in literature. Moreover, LOD and LOQ are comfortable lower than the maximum Cr allowed by Brazilian (25 µg g1
) and US US and European (1100 µg g-1) pharmacopeia.
(insert Table 2)
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3.4. Recovery tests and analytical results
To obtain a most reliable result, pharmaceutical drugs containing an external coating (PD11, PD12, PD13 and PD17) were grinded and sieved, as
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preliminary studies without sieving showed high values of RSD (> 30%). After
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this procedure, it was possible to achieve a better sample homogeneity, leading
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to more satisfactory RSD values (< 13%). Due to the lack of certified reference material for pharmaceutical drugs, the trueness of the proposed method was
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evaluated by comparing the results of the SS analysis with those obtained by analyzing the samples after an acid digestion. In this context, four drugs were
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analyzed by submitting them to a closed vessel microwave assisted acid digestion with (recovery test) and without analyte addition. The Cr
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concentrations obtained by SS analysis and in the digested samples were
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compared using the Student t-test (Table 3), with 95% of confidence level, and the results were in agreement. Moreover, these results, considering the
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investigated sample mass used in the acid digestion procedure (150 mg) and those employed in the direct solid sample analysis (7.5 mg maximum), confirm
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the samples homogeneity. The recovery values varied between 92 and 105% considering the four investigated samples. The precision of the result can be evaluated as the RSD, which was below 15% in all performed measurements (Table 3). Therefore, from the presented results for digested samples, recovery tests and RSD values, it is confirmed that the proposed method is accurate. After that, the developed method was employed for the pharmaceutical drugs and excipients analysis: eighteen pharmaceutical drugs (PD) and three
ACCEPTED MANUSCRIPT excipients (E) were analyzed. The results are shown in Table 3. The Cr concentration found are between 0.038 and 1.90 µg g -1. The results are within the stipulated values from Brazilian (25 µg g-1), US and EP (1100 µg g-1) [7-9].
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(insert Table 3)
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4. Conclusion
In this work it was developed an accurate, fast and reliable analytical method for
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the determination of Cr in pharmaceutical drugs and excipients by HR-CS GF AAS and direct analysis of solid samples. It avoided the intensive manipulation
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of the samples, reducing time and cost of the analysis besides the risk of sample contamination. The developed method allows the use of aqueous
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standards solutions for calibration, confirming that this method is simple and
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suitable for routine applications. All commercial analyzed samples showed Cr content much lower than values established by current Brazilian, US and
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European Pharmacopeias.
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Acknowledgements
The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their financial support. M.G.R.V. has scholarship from CNPq (grant no. 305679/2015-5) and W.B. (grant no. 1533/2013) and E.G.B. (grant no. 23038007479201144) from CAPES.
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ACCEPTED MANUSCRIPT Table 1. Graphite furnace temperature program for Cr determination in commercial pharmaceutical drugs by HR-CS SS-GF AAS. Temperature/ºC Ramp/ºC s-1
Stage
Hold time/s
90
30
20
Drying 2
120
10
20
Pyrolysis
1200
500
30
Atomization*
2600
3000
Cleaning
2650
1000
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*Argon flow rate was 2.0 L min-1 in all stages, except during the atomization,
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ACCEPTED MANUSCRIPT Table 2. Figures of merit for the determination of Cr in pharmaceutical drugs using HR-CS SS-GF AAS. Tpyr: 1200 °C and Tatom: 2600 °C Cr (357.868 nm)
Linear regression equation
A = 0.0021m (pg) + 0.0044
Correlation coefficient
0.9980
LOD (pg / ng g-1)
2.9 / 0.39a
LOQ (pg / ng g-1)
9.7 / 1.3a
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Parameters
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2.0 pg
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LOD and LOQ calculated for the maximum sample mass used: 7.5 mg.
ACCEPTED MANUSCRIPT Table 3. Cr concentration in pharmaceutical drugs (PD) and excipients (E) samples (n = 5). Solid samples RSD
Cr
RSD
(µg g-1 ± sd)
(%)
(µg g-1 ± sd)
(%)
PD1
0.307 ± 0.023
8
PD2
0.028 ± 0.004
14
PD3
0.134 ± 0.013
10
PD4
0.808 ± 0.113
14
PD5
0.042 ± 0.003
7
PD6
0.051 ± 0.005
PD7
0.088 ± 0.003
PD8
0.152 ± 0.014
10
PD9
0.083 ± 0.002
2
PD10
0.133 ± 0.015
10
0.093 ± 0.009
10
0.184 ± 0.024
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0.132 ± 0.004
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PD13
0.577 ± 0.050
9
PD14
0.038 ± 0.004
10
PD15
0.093± 0.009
10
PD16
0.170 ± 0.021
12
PD17
1.90 ± 0.20
11
PD18
0.145 ± 0.014
10
E1
0.315 ± 0.027
9
E2
0.072 ± 0.006
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0.085 ± 0.003
4
0.186 ± 0.010
6
1.96 ± 0.15
7
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4
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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1: Pyrolysis curves for Cr evaluating a (□) pharmaceutical drug (PD1) with the Aint values normalized to a sample mass of 0.5 mg and () a Cr (100 pg) aqueous standard solution. Tatom = 2600 ºC. Figure 2: Atomization curves for Cr evaluating a (□) pharmaceutical drug (PD1) with the Aint values normalized to a sample mass of 0.5 mg and () a Cr (100 pg) aqueous standard solution. Tpyr = 1200 ºC.
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Figure 3. Absorbance signal at (─) 2500 ºC and (--) 2600 ºC for chromium in (a) aqueous standard solution (100 pg) and (b) pharmaceutical drug (PD1).
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Figure 4: Time-resolved absorbance spectra of Cr for (a) PD7 (m = 0.719 mg, Aint = 0.1167) and (b) PD17 (m =0.108 mg, Aint = 0.4247) pharmaceutical drugs samples. Tpyr = 1200 °C and Tatom = 2600 °C.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
ACCEPTED MANUSCRIPT Highlights Metals contamination in pharmaceutical drugs and excipients. Chromium determination in pharmaceutical drugs. Use of direct analysis of solid samples by graphite furnace atomic absorption
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spectrometry A fast, reliable and simple method was developed for routine pharmaceutical
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No spectral interferences and symmetrical peaks were obtained.