Multivariate Approach in the Optimization Procedures for the Direct ...

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Multivariate Approach in the Optimization Procedures for the Direct Determination of Manganese in Serum Samples by Graphite Furnace Atomic Absorption Spectrometry Henrique José Ferraz Fabrino1, Josianne Nicácio Silveira2, Waldomiro Borges Neto1, Alfredo Miranda Goes3, Mark Anthony Beinner4, and José Bento Borba da Silva1,* 1Chemistry

Department, Federal University of Minas Gerais, Belo Horizonte, Av. Antônio Carlos no 6627; CEP 31270-901, Belo Horizonte—MG, Brazil; 2Laboratory of Occupational Toxicology, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Av. Antônio Carlos no 6627; CEP 31270-901, Belo Horizonte—MG, Brazil; 3Department of Biochemistry and Immunology, Federal University of Minas Gerais, Av. Antônio Carlos no 6627, CEP 31270-901, Belo Horizonte—MG, Brazil; and 4School of Nursing and Nutrition, Federal University of Minas Gerais, Av. Antônio Carlos no 6627; CEP 31270-901, Belo Horizonte—MG, Brazil

Abstract A method for direct determination of manganese (Mn) in human serum by graphite furnace atomic absorption spectrometry (GFAAS) was proposed in this work. The samples were only diluted 1:4 with nitric acid 1% (v/v) and Triton® X-100 0.1% (v/v). The optimization of the instrumental conditions was made using multivariate approach. A factorial design (23) was employed to investigate the tendency of the most intense absorbance signal. The pyrolysis and atomization temperatures and the use of modifier were available and only the parameter modifier use did not have a significant effect on the response. A Center Composed Design (CCD) presented best temperatures of 430°C and 2568°C for pyrolysis and atomization, respectively. The method allowed the determination of manganese with a curve varying from 0.7 to 3.3 µg/L. Recovery studies in three concentration levels (n = 7 for each level) presented results from 98 ± 5 to 102 ± 7 %. The detection limit was 0.2 µg/L, the quantifying limit was 0.7 µg/L, and the characteristic mass, 1.3 ± 0.2 pg. Intra- and interassay studies showed coefficients of variation of 4.7–7.0% (n = 21) and 6–8%(n = 63), respectively. The method was applied for the determination of manganese in 53 samples obtaining concentrations from 3.9 to 13.7 µg/L.

Introduction In humans and animals, manganese (Mn) is an essential nutrient and plays a role in bone mineralization, protein

* Author to whom correspondence should be addressed.

metabolism, metabolic regulation, cellular protection from free radical species, and synthesis of glycosaminoglycans (1). Mn can be a component of metalloenzymes, but must also be able to activate many enzymes with catalytic or regulatory function [i.e., transferase, decarboxylase, and hydrolase (1)]. Arginase, pyruvate carboxylase, and manganese-superoxide dismutase (MnSOD) can be cited among the enzymes containing Mn (1,2). The average blood concentration of manganese in normal adults is 9 µg/L (range 4–15 µg/L), according to Barceloux (3). After being absorbed, Mn is rapidly distributed to other tissues. In rats, the half-life in blood after intravenous injection of MnCl2 was estimated at 1.8 h (4). Considering the total organism, the biological half-life of manganese removal reported in the literature ranges from a shorter 13–43 days (5) to a longer period of 24–74 days (6). Occupational exposures reported cases of lead poisoning by Mn. Cases of neurotoxicity due to inhalation exposure have been diagnosed in workers at Mn dioxide mines (7), workers in dry cell battery factories (8), founders (9), and welders (10–12). Environmental exposure may occur due to the extraction, processing, and transportation of Mn ores; the use of organic pesticides containing Mn (13); use of the fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) (14–17); water intake containing high levels of Mn (14–18); and even by use of a street drug known as “Bazooka”, the cocaine-based drug contaminated with Mn-carbonate from free-base preparation methods (19). Mn toxicity has also been reported by ingestion in patients receiving long-term parenteral nutrition containing about 1 mg/day of parenteral Mn for adults or more than 40 µg/kg/day for children (20). Interestingly, Mn deficiency has

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also been noted in patients on total parenteral nutrition (21). Mn toxicity is evidenced primarily in the central nervous system and in lung tissue (following inhalation exposure), although cardiovascular, liver, reproductive, and developmental toxicities have also been noted (22). There is currently no established, reliable biological indicator (or biomarker) to evaluate Mn exposure. Some investigators suggest that Mn concentrations in blood seem to be fairly stable over long periods of time in humans exposed to this metal in mining and industrial environments, and thus can be used to reflect the Mn body burden (23,24). Others, mainly based on animal studies (25–27), point out that Mn is quickly eliminated from the blood circulation and possesses a rather short blood half-life, but a prolonged tissue half-life, following exposure. The discrepancy between blood and tissue half-life, and possibly a large tissue accumulation of Mn, may render the blood Mn level less relevant as an indicator of total body burden of Mn. Five days after the reported ingestion of 5– 10 g potassium permanganate crystals, the whole blood level was 1.63 mmol (90.5 mg) Mn/L, and 30 h later, the level fell to 0.76 mmol (42.2 mg) Mn/L (3). A recent study of welders conducted by this laboratory shows that career welders have a significantly higher serum Mn compared to control subjects; however, the elevated serum Mn concentrations among welders were not associated with welders’ length of employment. Thus, blood Mn may reasonably indicate recent, but not historical, exposure in welders (28). Thermodynamic models of Mn2+ in serum suggest that Mn exists in several forms, including species related to albumin (84%); as ion hydrated (6.4%); and in complex with bicarbonate (5.8%), citrate (2.0%), and other low molecular weight ligands (1.8%) (29). A similar model says that nearly 100% of Mn3+ is linked to serum transferrin (30,31). Mn2+ can be oxidized to Mn3+ which is very reactive and more toxic (32). The sample preparation required for trace analysis in complex matrices such as human serum may involve several stages, such as sample introduction using pneumatic nebulization, and may require a large sample because of the possibility of loss or contamination. The possibility of in situ digestion of samples, which requires little or no prior preparation, by use of the technique graphite furnace atomic absorption spectrometry (GFAAS), has been explored in several studies (33–39). The optimization of experimental parameters in univariate analysis by GFAAS (the chemical modifiers, pyrolysis time, pyrolysis, and atomization temperatures) requires a large number of time-consuming and costly experiments. Moreover, the interactions between optimized variables were not evaluated. Multivariate optimization seems to be adequate when a lot of variables are involved (40–42). The factorial design is a good and simple statistical tool that is used to verify the effects of variables and their interactions with few experiments (35–42). Center Composed Design (CCD) are possibly the most widely used classes of plans to fit quadratic models that may generate response surfaces for optimization of variables, which have been used as useful characteristics and provide good estimates for all coefficients, requiring fewer experiments, and provide conditions assessment and coefficients of the model, [i.e., regression and the lack of fit (38–42)]. In this work, a fac-

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torial and CCD design was employed to optimize the experimental conditions for the direct determination of manganese in diluted human serum samples by GFAAS with in situ matrix removal.

Experimental Instrumentation

All measurements were carried out with a SpectrAA Zeeman220 from Varian (Mulgrave, Australia), equipped with a graphite furnace, an autosampler (PSD – EL 98013384 ZC), and the polarized Zeeman background correction, all from Varian. A hollow cathode lamp for Mn from IST® (WL 22936, serial no. 12774, Imaging & Sensing Technology, Horseheads, NY), operating at 15 mA, with a slit width of 0.2 nm, and a wavelength of 279.5 nm. Argon 99.999% from Air Liquide® (Contagem, Brazil) was used as the purge gas with a flow rate of 3.0 L/min. Pyrolytic graphite coated tubes (63.100012-00, Varian) were used for all studies. Materials, reagents, and solutions

The following reagents were used: nitric acid (Suprapur®, 65%, part no. 1.00441.0250, Merck, Darmstadt, Germany); 1000 ± 2 mg/L stock manganese solution (Titrisol®, part no. 109988, Merck) in 5% (v/v) HNO3; 1000 mg/L iridium atomic spectroscopy standard solution (part no. 58195, Fluka, Buchs, Switzerland) in 1 mol/L hydrochloric acid; 1000 mg/L ruthenium standard solution (part no. 84033, Fluka) in 1 mol/L hydrochloric acid; 1000 mg/L rhodium ICP standard solution (part no. 1703450100, Merck) in HNO3 2–3%; 1000 mg/L tungsten standard solution prepared by dissolving 0.18 g of Na2WO4 (Merck) in 100 mL of water; and 1000 mg/L palladium atomic spectroscopy standard solution (part no. 76035, Fluka) in 1 mol/L HNO3. All of the solutions were prepared with deionized water with a specific resistivity of 18.2 mΩ cm–1 obtained from a Milli-Q system (Millipore, Bedford, MA) purifier immediately before use. Autosampler cups, tips for micropipette, and glassware materials were cleaned by soaking in 20% (v/v) HNO3 for least one day, rinsing many times with Milli-Q water, and drying. The autosampler washing solution, 0.2% (v/v) Triton® X-100 (Merck) plus 0.2% (v/v) nitric acid, was used to avoid analyte adsorption onto the surface of the container and clogging of the capillary sampling tip, as well as to improve the dispersion of the sample solution onto the wall graphite tube. A solution of Triton X-100 (Merck) 0.1% (v/v) with nitric acid 1% (v/v) in Milli-Q water was used as a diluent. Triton acts as a detergent to eliminate carbonaceous resides formed inside the graphite tube and also helps in the cleaning of the autosampler capillary between sampling. Sampling and sample treatment

Initially, blood samples were obtained by venipuncture from 53 adult volunteers using non-heparinized vacuum tubes (Vacuette, ref. 454092). This study was approved by the Research

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Ethics Committee (COEP) of UFMG (protocol number ETIC 523/07), and informed consent was obtained from each individual before blood collection. After collection, blood samples were subjected to centrifugation for 15 min at 3000 rpm, and serum samples were separated with the use of a pipette. Serum samples were stored in a freezer until ready to use. The only preparation of the samples was a simple 1:4 dilution with nitric acid solution 1% (v/v) with Triton X-100 0.1% (v/v), made directly in the autosampler cups of GFAAS. This solvent was chosen based on the good results obtained in previous works that used as diluent aqueous solutions of acid and surfactant at low concentrations (33–35), the amount of solutions pipetted into graphite tube was always 20 µL.

factorial design was constructed to evaluate, initially, the variables pyrolysis temperature [700°C (–) and 1200°C (+)], atomization [2000°C (–) and 2400°C (+)] and use of chemical modifier] tungsten (–) and untreated tube wall with permanent modifier (+)]. With the absorbance data obtained from these factorial design experiments, a Pareto chart was generated and from this, planning a CCD was carried out to determine the optimal values of pyrolysis and atomization temperatures. The pyrolysis temperature ranged from 419°C to 701°C, with a central point of 460°C, and the atomization temperature ranged from 2117°C to 2683°C, with a central point of 2400°C. In all experiments the temperature was kept clean at 2700°C for 2 s. Experimental data were processed using software Statistica 6.0 (42).

Graphite tube treatment

In the studies using a permanent modifier, the wall of graphite tubes were treated by applying 40 µL of 1000 mg/L of desired modifier and submitting the tube to a specific temperature program as published elsewhere (43–45). This procedure was repeated 13 times in order to obtain a deposit of 520 µg of permanent modifier, following by five injections of 40 µL of Milli-Q water to cleanse the graphite tube. Optimization strategy

A random serum sample was selected from the group of volunteers to be used in developing the method (reference sample). The reference sample, diluted 1:4 with nitric acid 1% (v/v) with Triton® X-100 0.1% (v/v), was contaminated with 0.2 µg/L Mn and used in optimization. Initially, the drying temperature and time were optimized from the conditions recommended by the manufacturer, without observing bubbling of the sample inside the graphite furnace. Readings were evaluated for height or peak area. Then, using temperatures and times of pyrolysis and atomization recommended by the manufacturer, we tested different graphite tubes to select two to present the best performances (higher absorbance with background corrected) for use in planning a factorial design, pipes with walls treated with permanent modifiers (tungsten, rhodium, ruthenium, niobium, titanium, and iridium), untreated tube walls with permanent modifier added with palladium (modifier solution), and tube walls with untreated and without using any chemical modifier. A 23 Table I. Optimized Furnace Program for Determination of Manganese in Human Serum Using GFAAS

Optimization procedure

The tubes with permanent tungsten modifier and without treatment with chemical modifier gave the highest absorbance readings with background correction, compared with other modifiers studied (section 2.5), and thus were used in the 23 factorial design. The Pareto chart (not shown), generated by factorial design shows that only the variable use of modifier had no significant effect on response (absorbance peak height) at 95% confidence level, and that the best results for the temperatures of pyrolysis and atomization were 700°C (–) and 2400°C (+), respectively. The variable primary use of chemical modifier was not a significant parameter, but the second order interactions were significant and showed better results with the use of W as permanent modifier. A CCD planning was conducted to determine the optimal temperatures for pyrolysis and atomization. The optimal values for the temperatures of pyrolysis and atomization were obtained by deriving the equation math (1): Abs = – 0.000002Ta2 – 0.0000005Tp2 – 0.0000001TaTp + 0.0102Ta + 0.0007Tp – 12.4282

Eq. 1

where Abs = absorbance; Ta = atomization temperature, and Tp = pyrolysis temperature. Even after reaching the optimum conditions, an undesirable accumulation of carbonaceous residues deposited on the tube wall after several heating cycles. To minimize the accumulation of this carbonaceous residue, 10 L of a solution of Triton X-100 0.1% were pre-injected, and submitting at the drying step. Table I shows the optimized furnace program.

Temperature (°C)

Ramp (s)

Hold (s)

Drying

110

5

40

3.0

Drying

140

10

30

3.0

Calibration

Pyrolysis

430

5

35

3.0

Pyrolysis

430

0

15

0.0

Aqueous calibration was compared with calibration curves derived from Mn-fortified serum samples (n = 3). The linear range studied was 0.7 to 3.3 µg/L with a coefficient of determination always greater than 0.99 for all curves. Statistical analysis (F tests and Student’s t-test) from the slopes of the curves showed a significant difference between them for the

Step

Ar Flow Rate (L/min)

Results and Discussion

Atomization

2568

0.9

2

0.0 (read)

Clean

2500

2

0

3.0

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range of 95%. Therefore, subsequent studies were done using calibration adjustment matrix. The linear regression equation obtained was Abs = (0.035 ± 0.002)CMn + (0.0004 ± 0.0002), as shown in Table II. Analytical figures of merit

The limit of detection (LOD) at the optimized conditions was 0.2 µg/L calculated according the IUPAC: 3 times the standard deviation for 10 distinct measurements of the blank, divided by the slope of the calibration curve. The quantifying limit (LOQ) found in this study was 0.7 µg/L and calculated 10 times the standard deviation for measurements of the blank, divided by the slope of the calibration curve. The characteristic mass of 1.3 ± 0.2 pg was obtained as an average of values found for each point on the curve (Table II). Luna and Campos (47) determined Mn in urine samples by GFAAS making use of Pd (NO3)2 as modifier with LOD of 0.6 µg/L. Pinto et al. (48) reported the direct determination of Mn in urine and whole blood employing GFAAS using graphite tubes without modifier; the characteristic masses found were 0.47 and 1.81 pg, and the detection limits were 0.2 and 0.3 µg/L for urine and whole blood, respectively. In another study (49) Table II. Analytical Figure of Merits for the Determination of Manganese in Human Serum Under Optimized Experimental Conditions Using GFAAS Parameter

Results

Regression equation (n = 3)

Abs = (0.035 ± 0.002) CMn + (0.0004 ± 0.0002)

R2 (n = 3)

0.993 ± 0.002

Linear range (µg/L)

0.7–3.3

LOD (µg/L)

0.2

LOQ (µg/L)

0.7

Characteristic mass (pg, n = 6)*

1.3 ± 0.2

Determination of manganese in human serum samples

The manganese concentrations in the sera of 53 volunteers were determined using the optimized experimental conditions. Table IV presents the levels of manganese in the samples. The levels of Mn found in the serum of participants of this study included a range of 3.9 to 13.7 µg/L, with median concentration of 7.1 µg/L, which are values considered normal for the population free of this metal contamination. Röllin et al. (52) analyzed the level of Mn content in the blood samples of the children in Johannesburg and in Cape Town using GFAAS obtained results of median of 9.2 and 6.2 µg/L for the two regions, respectively. In other work, the whole blood and serum Table IV. Manganese Concentration in 53 Samples of Human Serum Analyzed by the Proposed Method Mn Mn Mn Concentration Concentration Concentration Sample (µg/L) Sample (µg/L) Sample (µg/L)

* Characteristic mass recommended by the manufacturer for the determination of Mn in water = 0.6 pg.

Table III. Results of Precision and Accuracy of the Method Developed for Manganese Determination in Human Serum Samples Mn Concentration (µg/L)

Intraassay CV (%, n = 7)

Interassay CV (%, n = 21)

Recovery (%, n = 7)

0.6 1.8 2.4 Average

4.9 4.7 7.0 5.5 ± 1.3

8.0 6.0 6.1 6.7 ± 1.1

98 ± 5 100 ± 5 102 ± 7 100 ± 6

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simultaneous determination of As, Mn, Co, and Ni in urine employing GFAAS, Pd was used as chemical modifier, obtaining LOD for Mn of 0.22 µg/L. The intraassay precision was evaluated by relative standard deviation (RSD%) of serum added to 0.6, 1.8 and 2.4 µg/L of Mn (n = 7, distinct replicates prepared as described in item 2.3) and interassay precision by RSD %, and also seven different replicate concentrations of the same, but on three different days (n = 21, for each concentration). The band found the relative standard deviation for the precision intra- and interassay were 4.7% to 7.0% and 6% to 8%, respectively (Table III). The results are in accordance with the criteria established by The International Association of Official Analytical Chemistry (50); the coefficient of variation (RSD) might vary from 15% to 30%, depending on the concentration range used (1–100 µg/L). The accuracy was evaluated by recovery studies in serum samples added 0.6, 1.8, and 2.4 µg/L of Mn. The recovery values ranged from 98 ± 5% to 102 ± 7% (Table III), respecting the limit satisfactorily included in the range 80% to 120% (51) and showing good accuracy of the method.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

13.7 ± 0.1 5.1 ± 0.0 11.1 ± 0.1 8.8 ± 0.1 7.2 ± 0.1 6.0 ± 0.2 7.9 ± 0.1 11.9 ± 0.4 6.4 ± 0.2 9.9 ± 0.3 4.5 ± 0.2 3.9 ± 0.2 6.0 ± 0.2 6.0 ± 0.2 9.2 ± 0.2 10.5 ± 0.1 4.8 ± 0.3 12.6 ± 0.0

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

9.7 ± 0.1 8.3 ± 0.1 10.2 ± 0.4 5.9 ± 0.5 5.0 ± 0.1 4.9 ± 0.1 5.2 ± 0.3 9.4 ± 0.2 9.5 ± 0.1 8.1 ± 0.1 6.5 ± 0.4 7.1 ± 0.4 5.0 ± 0.2 5.7 ± 0.0 6.5 ± 0.3 4.9 ± 0.1 6.0 ± 0.3 7.8 ± 0.3

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

4.5 ± 0.0 7.3 ± 0.4 7.5 ± 0.2 6.5 ± 0.3 6.4 ± 0.4 7.1 ± 0.2 6.4 ± 0.5 5.4 ± 0.3 8.8 ± 0.3 5.6 ± 0.1 8.9 ± 0.4 5.0 ± 0.2 4.1 ± 0.0 7.9 ± 0.4 7.7 ± 0.6 9.0 ± 0.3 8.1 ± 0.3

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manganese concentrations measured by GFAAS showed average values between 9.03 and 1.82 µg/L, respectively (53).

Conclusions The method requires a minimum procedure in sample preparation, involving a simple sample dilution Triton X-100 0.1% (v/v) in 1% (v/v) nitric acid and removal of the matrix in situ with a permanent modifier tungsten, helping to minimize the analytical time and cost. Use of the factorial design fragmental followed by the CCD design is a simple and fast procedure to evaluate the GFAAS analytic conditions for manganese determination in diluted serum samples. The response surface analysis revealed a maximum absorbance. A pre-injection of a solution of Triton X-100 0.1% (v/v) and drying of this solution, ensured a more efficient cleaning of the pipe and with the contribution of the permanent modifier, a longer cycle of firing was reached. Obtained figures of merit are very adequate for GFAAS determinations. The simplicity of the method of preparation, as well as the set of instrumental and analytical conditions, permitted an adequate determination of manganese in human serum samples.

9. 10. 11. 12. 13.

14. 15.

16. 17. 18.

Acknowledgments 19.

The authors wish to thank the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil.

20. 21.

22. 23.

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Manuscript received January 24, 2011; revision received May 5, 2011.