Stepwise injection spectrophotometric determination of cysteine in biologically active supplements and fodders

Microchemical Journal 110 (2013) 369–373

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Stepwise injection spectrophotometric determination of cysteine in biologically active supplements and fodders Andrey V. Bulatov a,⁎, Anastasiia V. Petrova a, Andriy B. Vishnikin b, Leonid N. Moskvin a a b

Department of Analytical Chemistry, Faculty of Chemistry, Saint-Petersburg State University, Saint-Petersburg, prosp. Universitetskij 26, 19850, Russia Department of Analytical Chemistry, Faculty of Chemistry, Oles Gonchar Dnipropetrovsk National University, Gagarina 72, Dnipropetrovsk 49010, Ukraine

a r t i c l e

i n f o

Article history: Received 24 January 2013 Received in revised form 23 April 2013 Accepted 23 April 2013 Available online 9 May 2013 Keywords: Stepwise injection analysis Flow-batch analysis Spectrophotometry Cysteine

a b s t r a c t A stepwise injection analysis system was developed to monitor the concentration of L-cysteine in biologically active supplements (BAS) and fodders. It is based on the rapid redox reaction of L-cysteine with 18-molybdo2-phospahte heteropoly anion (18-MPA) and the detection of the formed heteropoly blue with a spectrophotometry. The method has relatively wide optimal ranges of pH and 18-MPA concentration −5.0 to 7.3 and 0.4 to 2.0 mM, respectively. Under optimized operating conditions the performance of the SWIA system was linear up to a concentration of L-cysteine of 0.1 mM (R2 = 0.994) with a detection limit of 0.003 mM and a sample frequency of 20 h−1. The SWIA system was employed to determine the concentration of L-cysteine in BAS and fodder. The obtained data were in good agreement with those measured by a HPLC method. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The biologically active food supplement market has been one of the fastest growing segments in the last several years. The number of registered biologically active food additives in Russia is now over 2000. The attractiveness of biologically active food supplements is due to the fact that they are relatively easy to develop and launch into production. This is one of the reasons why new analytical methods for their control have to be developed. Cysteine and its salts are widely used as supplements (E 920) in food and agricultural products. Cysteine is a non-essential α-amino acid containing nonpolar sulphydryl (―SH) group that provides its participation in a variety of biochemical reactions. The sulphydryl group can be easily oxidized to form disulfide bonds that can be converted back to corresponding thiols through enzymatic activities and plays important structural roles in proteins [1]. The lack of cysteine is responsible for many kinds of different diseases. It causes slow hair growth, depigmentation, damage of liver and muscles. Increased levels of cysteine lead to the disturbance of normal brain function [2]. The methods reported up to now for the determination of cysteine include batch spectrophotometric [3–7], fluorimetric [8,9], chemiluminescence [10–12], HPLC [13–16], capillary electrophoresis [17,18], and electrochemical procedures [19–23]. These methods are generally laborious and time consuming. In addition, chromatographic procedures require expensive and complicated instrumentation that make them unattractive to routine analysis. For the FIA/SIA determination of cysteine different detection techniques, including electrochemical [24,25], ⁎ Corresponding author. Tel.: +7 91126113385; fax: +7 812 372 44 21. E-mail address: [email protected] (A.V. Bulatov). 0026-265X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.microc.2013.04.020

chemiluminescent [10,26], enzymatic [27,28] and spectrophotometric [29–33] were used. Lately, the significance of the Wells–Dawson heteropoly anion 6− (18-MPA) for the deter(HPA) 18-molybdo-2-phosphate P2Mo18O62 mination of a number of reducing agents in batch and sequential or stepwise injection systems was shown [34–37]. It should be noted that the history of the application of HPAs in analysis began with intensive use of complexes having Wells–Dawson structure in biochemical analysis. The 18-tungsto-2-phosphate HPA (Folin uric acid reagent, phospho-18-tungstic acid) was for a long time one of the main reagent for the determination of cystine and cysteine [38–42]. Flow analysis techniques are the well-established analytical tool for solving problems of routine analysis and quality assurance control. Especially in quality control of pharmaceuticals or other samples with simple matrix such as biologically active supplements, flow analysis techniques with spectrophotometric detection can easily and effectively replace complicated and expensive chromatographic separation methods since the measured active ingredient usually exists in high concentrations and the common excipients cause no serious interferences. Stepwise injection analysis (SWIA) is one of the universal solutions for the automation of analytical reactions in which the equilibration in the reaction is reached but dispersion of the reactants is prevented [36,43]. SWIA manifold is a hybrid analyzer exploiting characteristics of both flow and batch systems. It combines the advantages of automated control of flows such as high throughput, complete and precise control of reactant volumes and timings of operations, low cost, low consumption of the reagents and low effluent production with the flexibility and the versatility of mixing chamber (MC). The scheme of the SWIA manifold is close to that for flow-batch analysis (FBA) system [44]. At the beginning of each analytical cycle,

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the sample solution as well as certain volumes of all of the reagent solutions specified by analytical procedure is sequentially supplied with the use of a peristaltic pump through corresponding channels of multiway valve into MC. As opposite to this, a multicommutation system consisting usually of three-way solenoid valves is built into the FBA manifold for directing the fluids towards the MC. On the other hand, similar to the SIA setup, in SWIA all chemistries are applied through a single-channel manifold. MC is the main component of the SWIA manifold providing intense and effective mixing of a reagent and sample solutions by the gas flow. The reaction mixture is kept in MC the time needed for the completion of all of the chemical and physical processes, including mixing, chemical reaction, extraction, etc. Magnetic stirrer is generally used in FBA systems for mixing but it complicates the configuration. In addition, using the MC greatly simplifies the execution of some analytical operations. For instance, gaseous samples can be analyzed without inclusion into the scheme of flow system any additional absorption devices [45–48]. The contents of the mixing (reaction) chamber are pumped through the flow detector using the reversible pump and directed to waste. All the instrumental parameters of SWIA manifold are computer controlled. We have found that intensively colored heteropoly blue is formed rapidly and selectively in the reaction between cysteine and 18-MPA. The aim of this study was to develop simple, fast, and robust automated SWIA spectrophotometric method for the determination of cysteine in biologically active supplements and fodders which allows selective and sensitive determination of the analyte in a wide concentration range with minimum sample pretreatment and low operational cost. 2. Experimental 2.1. Reagents Ammonium salt of 18-molybdo-2-phosphate HPA (NH4)6P2Mo18 O62× 14H2O was synthesized according to the procedure described in Ref. [36]. About 0.01 M solution of 18-MPA was prepared by dissolving 0.7855 g of the synthesized salt and diluting to 25 mL with distilled water. The stock solutions of 0.01 M cysteine chloride hydrate (Senn Chemical, Switzerland) was daily prepared by dissolving accurately weighed amounts in 0.01 M HCl solution and stored in a refrigerator. The acetate buffer solution of pH 5.0 was used for adjusting the pH of the samples. All chemicals were of analytical-reagent grade. 2.2. Apparatus The absorbance was measured by means of SHIMADZU UVmini-1240 (Shimadzu Scientific Instruments, Kyoto, Japan) spectrophotometer

equipped with a standard quartz cells with an optical path length of 10 mm. The pH of solutions was measured by an I-500 potentiostat (Akvilon, Russia) using glass indicator electrode and Ag/AgCl as a reference electrode. The SWIA manifold (Fig. 1) was based on a flow analyzer PIAKON30-1 (Rosanalit, Saint-Petersburg, Russia). It included a bidirectional peristaltic pump ensuring a reverse flow, a six-port titanium valve, a mixing chamber which had cylindrical shape and was funnel-shaped at the bottom (glass tube 350 mm in height and 10 mm in inner diameter), a spectrophotometric detector with flow cell (optical path length of 5 cm), and communication tubes (PTFE, 0.5 mm in inner diameter). The analyzer was operated automatically by means of a computer. 2.3. Procedure for the SWIA determination of cysteine According to the scheme of stepwise injection analysis (Fig. 1), at the first step, 0.2 mL of sample solution, 0.2 mL of acetate buffer solution (pH 5.0) and 0.2 mL of 4 × 10−4 M solution of 18-MPA were sequentially passed through multiway valve by means of peristaltic pump into MC. After that, the flow of argon gas was introduced into the system to mix reaction mixture in MC. The flow rate was maintained at 3.5 mL min−1. After 60 s, the solution from MC was passed through port e by the reverse flow to the flow cell of spectrophotometric detector. Absorbance of analytical form was measured in stopped-flow mode. After measurement of analytical signal, the solution was passed away. At the next step, the washing with distilled water of hydraulic communications of the manifold and MC was performed. The measurement of analytical signal for blank solution was carried out by the above mentioned algorithm. But in this case instead of the sample solution, distilled water was passed through the port f. To provide the automated control of sequence and duration of all stages of analysis by personal computer, the operating program was written (Table 1). 2.4. Sample preparation For the analysis of biologically active supplements, the content of one capsule was ground, then 0.35–0.40 g of obtained powder was dissolved in distilled water, transferred to a 25 mL volumetric flask, and the volume was adjusted to the mark with distilled water. The solution was then centrifuged at 5000 rpm for 15 min and filtered through a 0.45 μm membrane filter for separation of insoluble components of biologically active supplements. Before analysis, sample was tenfold diluted two or three times and appropriate aliquot of this solution was used for the analysis. Sample preparation for the analysis of fodders included an acidic hydrolysis of samples [49,50]. The amount of sample (0.35–0.40 g)

Fig. 1. The scheme of the manifold for SWIA determination of cysteine.

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Table 1 The sequence of steps in the optimized control program for SWIA determination of cysteine in biologically active supplements and fodder. Time, s

Volume, mL

Valve position

Direction of flow

10 10 10 60 35 20 20 40

0.2 0.2 0.2 – 0.6 – 0.4 0.4

a b c d e c d c

+1 +1 +1 +1 −1 0 −1 +1

a

a

Measurement of the absorbance

Description of the operation

Off Off Off Off On On Off Off

Filling of MC with sample solution Supply of buffer solution into MC Supply of 18-MPA solution into MC Stirring of the reaction mixture with argon Moving of the reaction mixture into the flow cell Absorbance measurement Washing of the MC with distilled water Washing of the flow cell with buffer solution

−1 and +1 refer to the clockwise and counterclockwise rotation of the pump, respectively; 0 — no rotation.

and 10 mL of 6 M HCl were put in vials with hermetically screwed heads. After stirring for 5 min, the vials were hermetically locked and placed in thermostat. The hydrolysis proceeded at 110 °C for 30 min. After filtration through glass filter, 5 mL of filtrate was placed into volumetric flask (25 mL) and filled with distilled water. Then the determination of cysteine was performed according to the developed SWIA procedure.

process for 18-MPA reduction. The first stable reduction product for 18-MPA is two electron heteropoly blue having molar absorptivity of 1.15 × 10 4 mol L −1 cm −1 [35]. In the excess of cysteine four electron heteropoly blue is formed with an absorption maximum at 690 nm (ε = 2.3 × 10 4 mol L −1 cm −1).

2.5. Procedure for the determination of cysteine by a reversed-phase HPLC method

Optimization of SWIA manifold parameters is much simplified comparing with that for FIA/SIA methods. Almost all of the optimal parameters found for batch method can be directly used, including the concentration of the reagent, pH, and reaction time, thus allowing to avoid time-consuming, complex, and sometimes troublesome multivariate optimization. The ranges of optimal conditions are sufficiently broad and remain the same as in batch method, thus positively influencing the robustness of the analytical procedure. The parameters of the SWIA manifold were optimized with the objective of achieving maximum reproducibility and selectivity with the current spectrophotometric method so that it could be applied to the analysis of cysteine in biologically active supplements and fodders (Table 2). The most important parameter defining the selectivity and completeness of the reaction between cysteine and 18-MPC is acidity. The dependence of analytical signal on pH was studied in SWIA conditions. The measured absorbance was maximum and stable in the range of pH from 5.0 to 7.3 (Fig. 2). At pH > 8 basic hydrolysis of 18-MPA proceeds very quickly that probably hinders the complete oxidation of cysteine, while at pH b4.5 reduction process becomes too slow. For further experiments, an acetic acid–sodium acetate buffer solution with pH 5.0 was used for the acidification because at this pH influence of interfering species (e.g. pyridoxine) was the least. In addition, in the chosen conditions stability of 18-MPA is maximum. The absorbance of the obtained heteropoly blue is stable during at least 1 hour. Only slight excess of 18-MPA above the theoretical amount is necessary for the complete formation of heteropoly blue. According to the obtained data (Fig. 3), analytical signal is maximum beginning

The results for the determination of cysteine in fodders and biologically active supplements by SWIA method were compared with those obtained by means of phenylisothiocyanate derivatization, HPLC reverse-phase separation, and ultraviolet detection at a wavelength of 254 nm. HPLC analysis was carried out on a Shimadzu LC-20 Prominence liquid chromatograph (Shimadzu Corporation, Kyoto, Japan) with UV detection (254 nm). The chromatographic separation was achieved by Supelco C18 HPLC column (250 × 4.6 mm, 5 μm particles size) in a gradient elution mode. The eluent flow rate was set at 1.2 mL min −1 and the column was thermostated at 40 °C. A mixture containing 6.0 mM sodium acetate solution of pH 5.5 and 1 % isopropanol in acetonitrile was used as a mobile phase. For the preparation of calibration curve 15, 25, 50, 100, and 150 μL of 0.01 M cysteine solution were placed in 5 tubes and dried in air flow at 65 °C. Then 0.1 mL of 0.15 M NaOH was added and the solution was thoroughly mixed. After that, 0.35 mL of 5% phenyl isothiocyanate in isopropanol and 0.5 mL of distilled water were added. The solution was dried at 60 °C for 15 min. Solid residue was dissolved in 1 mL of distilled water, filtered and injected into the HPLC system. 3. Results and discussion 3.1. Color reaction The reaction between cysteine and 18-MPA occurs very rapidly in a wide range of pH and is accompanied by the formation of intensely colored reduced form of heteropoly complex. In the sufficiently high excess of the reagent, reaction goes in accordance with the following equation:

The absorbance spectrum of the reduced form of heteropoly complex in visible region has only one absorption maximum situated at 820 nm. Corresponding molar absorptivity for cysteine was equal to 5.9 × 10 3 mol L −1 cm −1 proving the fact that the stoichiometry of the reaction between cysteine and 18-MPA is 2:1. Calculations consider a one electron process for cysteine oxidation, and a two electron

3.2. Optimization of SWIA manifold parameters

Table 2 Figures of merit for cysteine determination by the proposed SWIA method. Parameter

SWIA method

pH 18-MPA concentration (mM) Volume of sample (μL) Volume of 18-MPA reagent (μL) Time of stirring with argon in MC (s) Flow rate through the detector (μL s−1) Linear range (μM) Calibration graph Intercept Slope (L mol−1) Correlation coefficient Detection limit (μM) Quantification limit (μM) Sampling frequency (per hour)

5.0 0.4 200 200 60 30 10–100 0.023 6880 0.994 3 10 20

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Fig. 2. Dependence of the absorbance of heteropoly blue formed in the reaction between cysteine and 18-MPA on the pH of the solution. C(Cys) = 10−4 М, C(18-MPA) = 4 · 10−4 М, λ = 820 nm, l = 1 cm.

from 0.4 mM of 18-MPA. This concentration was chosen as optimal being a compromise between complete formation of heteropoly blue and minimal consumption of the reagent. The extent of transformation of cysteine to cystine depends on the reaction time and efficiency of reactant mixing in MC. Dependence of the absorbance of heteropoly blue on the mixing time of reactants by flow of gas in MC is represented in Fig. 4. Irrespective of nature of gas used for stirring, absorbance has stopped to grow after 60 s of mixing. Nevertheless, using the stirring with an inert gas has to be preferred because greater absorbance is achieved in this case. This result can be easily interpreted when the ability of cysteine or heteropoly blue to oxidation by oxygen present in air will be taken into account. Flow rate of gas supplied by means of peristaltic pump through port d of multiport valve into MC was set at 3.5 mL min −1 thus attaining better throughput.

Fig. 4. Influence of the mixing time of reactants in MC and the nature of gas used for the mixing on the absorbance of heteropoly blue. C(Cys) = 10−4 M, C(18-MPA) = 4 · 10−4 M, pH = 5.0, λ = 820 nm, l = 1 cm.

excipients was studied. Increasing concentrations of the possible interfering agents were added to a solution mixture with a fixed amount (40 μM) of cysteine, and the corresponding analytical signals were recorded. The tolerance limit was defined as the maximum concentration of potential interfering substance that caused a change in the signal of ±5%. The results are summarized in Table 3. No interference was found from common inorganic cations and anions up to 0.1 M. The effect of oxidative properties of Cu(II) and Fe(III) becomes apparent after 0.8 mM. Interference from glutathione and ascorbic acid is significant already at 0.02 mM; at the same time salicylic acid, tartaric and citric concentrations of 7 mM have no influence. All the amino acids studied except cysteine do not interfere with the determination of cysteine by the proposed method. 3.4. Calibration curve, sensitivity, precision, and application

3.3. Effect of coexisting foreign substances To assess the selectivity of the developed SWIA method, the effects of typical interferents present in biologically active supplements and fodders were investigated. The influence on the cysteine determination of some compounds possessing reducing properties, including those having phenolic, carboxylic, and hydroxylic groups, as well as

The analytical signal measured as a difference between absorbances for analyte and blank solutions was linear in the range of concentrations 10–100 μM for cysteine. The detection limit defined as three times the standard deviation of the blank was 3 μM. The reproducibility of the proposed procedure and sample throughput were determined by repeated injections of a sample containing 10 and 50 μM of cysteine. The relative standard deviations were 0.9% and 1.0% (n = 10), respectively, and the sample throughput was 20 measurements per hour. Other parameters of the calibration curve are listed in Table 2.

Table 3 Influence of some interfering species on the determination of 4 × 10−5 M of cysteine.

Fig. 3. Dependence of the absorbance of heteropoly blue formed in the reaction between cysteine and 18-MPA on the concentration of 18-MPA. C(Cys) = 10−4 М, pH = 5.0; λ = 820 nm, l = 1 cm.

Species

Tolerable concentration (M)

Na+, K+, Mg2+, Ca2+ NO3–, SO42–, PO43–, Cl– Cu2+, Fe2+ Na2SO3 Na2S2O3 KNaC4H4O6 Saccharose, paracetamol Uric acid Citric acid Salicylic acid, acetylsalicylic acid Norepinephrine, epinephrine, methyldopa Ascorbic acid, glutathione Lysine, proline, serine, tyrosine, glycine, alanine, valine, methionine, glutamic acid

0.1a 0.1 a 8 × 10−4 7 × 10−3a 3 × 10−3a 7 × 10−3a 0.01 a 1 × 10−4 7 × 10−3a 7 × 10−3a 2 × 10−4 2 × 10−5 1 × 10−3a

a

Highest studied concentration of the interferent.

A.V. Bulatov et al. / Microchemical Journal 110 (2013) 369–373 Table 4 Determination of cysteine in biologically active supplements and fodder using the proposed method and the HPLC reference method (mg of cysteine/capsule ± confidence intervals of the method for n = 5 and 95% confidence level). Samplea

SWIA method

Reference HPLC method [50]

t-Test

«Fitoval» (KRKA) «Revalid» (TEVA) Fodder

112 ± 15

108 ± 9

0.63

55 ± 5

53 ± 4

0.86

1.22 ± 0.08 g/kg

1.15 ± 0.08 g/kg

1.72

a

Composition of samples: Fitoval (KRKA) 200 mg medicinal yeast, 100 mg L-cysteine, 35 mg calcium pantothenate, 2 mg thiamine, 2 mg riboflavin, 2 mg pyridoxine hydrochloride, 0.1 mg biotin, 2 mg cyanocobalamin, 0.2 mg folic acid, 10 mg iron, 5 mg zinc, 1 mg cooper; Revalid (TEVA) 100 mg methionine, 50 mg L-cysteine, 50 mg calcium pantothenate, 1.5 mg thiamine hydrochloride, 10 mg pyridoxine hydrochloride, 20 mg p-aminobenzoic acid, 50 mg golden millets extract, 50 mg wheat-germ extract, 50 mg medicinal yeast, 2 mg iron, 2 mg zinc, 0.5 mg copper, the colors E 104 (Quinoline yellow) and E 132 (Indigo blue) and excipients.

The developed SWIA as well as HPLC method were applied to the determination of cysteine in real samples. Two samples of biologically active supplements were commercially obtained at a local drug store, including «Fitoval» (KRKA, Slovenia) and «Revalid» (TEVA, Israel) in capsules containing 100 and 50 mg of cysteine, respectively. Samples of cattle fodder were used for the analysis. The data in Table 4 clearly indicate that the cysteine contents, as measured by the proposed SWIA method, were in good agreement with those obtained by the HPLC method. The Student's t-test was used to compare the average contents of cysteine. The calculated value of t in all the cases was less than the critical value of t (2.31) at P = 0.95 and 8 degrees of freedom, thus showing the absence of significant differences between the average cysteine concentrations found by the SWIA and HPLC methods (Table 4). 4. Conclusions A very simple and robust spectrophotometric SWIA method for the determination of cysteine concentration has been developed by using as photometric reagent 18-molybdo-2-phosphate heteropoly anion. Reaction is fast, sensitive and highly reproducible. The proposed method combines full automation of the analytical procedure with a simple manipulation of a reagent solutions, and sufficiently high throughput of 20 determinations/hour. The method has relatively wide optimal ranges of pH and 18-MPA concentration − 5.0 to 7.3 and 0.4 to 2.0 mM, respectively. The used reaction has high selectivity allowing the determination of cysteine in biologically active supplements and fodders. The application was verified by the determination of cysteine by HPLC method. Acknowledgement This work was supported by the Russian Foundation for Basic Research (project no. 13-03-00031-a) and the Ministry of Education and Science of the Russian Federation for the financial support in form of Scholarship of the Russian President (the order no. 539).

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