Anal Bioanal Chem (2002) 374 : 466–476 DOI 10.1007/s00216-002-1497-2
O R I G I N A L PA P E R
P. Moreno · M. A. Quijano · A. M. Gutiérrez · M. C. Pérez-Conde · C Cámara
Stability of total selenium and selenium species in lyophilised oysters and in their enzymatic extracts
Received: 15 March 2002 / Revised: 20 June 2002 / Accepted: 10 July 2002 / Published online: 14 September 2002 © Springer-Verlag 2002
Abstract To obtain reliable information on speciation analysis it is necessary to previously evaluate the stability of the species in the sample of interest. Furthermore, in those cases in which sample treatment to extract the species is time-consuming, an evaluation of how to maintain species integrity in the extracts is paramount. Thus, the present paper reports the stability of total Se, SeMet and TMSe+ in freeze-dried oyster and in the enzymatic extracts stored in Pyrex and polyethylene containers at different temperatures (–18, 4 and 20 °C). Total selenium determinations and Se speciation were carried out by HG-AAS after acid digestion in a microwave oven and by on-line coupling of cation exchange HPLC-ICP-MS after enzymatic hydrolysis, respectively. The results obtained for the freeze-dried sample showed that total Se and the selenium species evaluated are stable for at least 12 months, under all the conditions tested. However, Se species in the enzymatic extracts are only stable for 10 days if stored at 4 °C in Pyrex containers. These results show that the extracts do not necessarily have to be analysed just after sample treatment. Keywords Selenium species · Stability · HPLC-ICP-MS
Introduction Selenium is both an essential and a toxic trace element for living organisms in a narrow range of concentration. Low levels of selenium are necessary for human metabolism, but higher concentrations of this element may cause
P. Moreno · A.M. Gutiérrez (✉) · M.C. Pérez-Conde · C. Cámara Departamento de Química Analítica, Facultad de C.C. Químicas, Universidad Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain e-mail:
[email protected] M.A. Quijano Departamento de Ingeniería Civil, E.U.I.T. de Obras Públicas, Universidad Politécnica de Madrid, Alfonso XII 3 y 5, 28014 Madrid, Spain
health damage. As its nutritional bioavailability and toxicity have been found to be dependent on the concentration level and the chemical form ingested [1, 2, 3, 4], it is important to determine the selenium species in food, especially in seafood, because of its bioaccumulation capacity. Several forms of selenium have been reported to exist in the diet. In animal foods and plants almost all of the selenium is protein bound [5, 6] as selenoproteins containing seleno amino acids, such as selenocysteine (SeCys) [7, 8] and selenomethionine (SeMet) [8, 9, 10]. The trimethylselenonium ion (TMSe+) is known to be a detoxified form of more toxic selenium compounds [11, 12] and has been identified in previous works on samples of tuna and mussel [13] and oyster [14]. Evaluation of the species stability under several conditions before their determination is a necessary preliminary step. The storage conditions may affect the accuracy of the results for various reasons, such as species interconversion, volatilisation, adsorption, precipitation, interaction with the container material, microbial activity, temperature, pH, light action, etc. [15, 16, 17]. Some studies have been performed on the stability of selenium species in aqueous solutions [18, 19]. The best storage conditions for organic selenium species in these solutions were found to be Pyrex containers at 4 and –20 °C in the dark. The authors observed excellent stability of TMSe+, SeMet and selenocystine (SeCys2) for 3 months under all the conditions tested. Inorganic selenium species were found to be stable, without need to acidify the samples, for 12 months in polyethylene and PTFE containers at –20 °C [18]. However, little information is available about the stability of selenium species in more complex matrices. Only a few studies have been performed on biological fluids such as urine [17, 20]. On the other hand, a few stability studies have been performed on alkylated selenium species. These compounds are volatile so losses by volatilisation may occur, even at room temperature, within 1 day when airtight containers are used [21]. Two factors greatly influence selenium species stability: the concentration level present in the sample and the
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matrix. For example, aqueous solutions of SeMet with concentrations ranging from 10 to 100 µg L–1 in acidified high ionic strength medium, are stable for 120 days when stored in glass and polyethylene containers at 4 and –20 °C. However, significant losses were observed for concentrations lower than 10 µg L–1 in glass and polyethylene containers in a low ionic strength matrix [22]. Therefore, as it has been reported that selenium species stability is dependent on the matrix, concentration level and the treatment followed for the sample analysis, it is necessary to perform stability studies for each case. Several procedures based on proteolytic hydrolysis have been developed to release Se compounds from proteins: treatment with tetramethylammonium hydroxide [10]; acid hydrolysis performed in HCl and anaerobic medium [23, 24]; and enzymatic hydrolysis [13, 14, 25, 26]. The latter is considered the most promising process for solid biological samples. Selenium speciation methods with low detection limits are usually required when biological samples are analysed. Extensive information on analytical techniques for selenium speciation can be found in several reviews [11, 12, 13, 14, 27, 28, 29, 30, 31, 32], which indicate that the most common analytical detector employed is inductively coupled plasma mass spectrometry (ICP-MS) coupled with chromatographic systems [11, 12, 13, 14, 31, 32, 33, 34, 35, 36, 37, 38]. This work focuses on the stability of total selenium and selenium species, both in lyophilised oysters and in their enzymatic extracts. The stability tests on the enzymatic extracts obtained from lyophilised oyster allow to know for how long the integrity of the extracts can be maintained.
Experimental Instrumentation Lyophilised sample was digested for total Se determination in doubled-walled composite vessels (ACV) using a 1000 W MSP (Microwave Sample Preparation system) microwave oven (CEM, Mattheus, NC, USA). A 2380 Perkin Elmer (Norwalk, CT, USA) atomic absorption spectrometer was used to determine the total selenium content. The continuous manifold used to generate the selenium hydride was based on the use of a four-channel peristaltic pump (Gilson HP4), a mixing and reaction coil (0.5 mm i.d. Teflon tubing) and a U-tube gas-liquid separator. An argon stream transferred selenium hydride to the quartz cell, which was electrothermically heated at 900 °C. Detection by ICP-MS was carried out in a VG Instrument PQ3 (Thermo Instruments, Uxbridge, Middlesex, UK). Before coupling the chromatographic system, the ICP-MS working conditions (torch box position, lens, quadrupole resolution, Ar flows) were optimised. A standard solution was used that contained elements spanning the mass range from beryllium to uranium, at a concentration of 10 µg L–1 for ICP-MS general checking. Then, the nebulization Ar pressure and lens voltages were readjusted with a 20 µg L–1 Se standard solution, in order to obtain the best signal/noise ratio for this element. The chromatographic system consisted of a Milton Roy CM4000 HPLC pump (Milton Roy LDC Division, Riviera Beach, FL, USA) with a Rheodyne (Rohnert Park, California, USA) six-port sample injection valve fitted with a 100 µL loop. Separations were carried
out in a Hamilton PRP-X200 (10 µm, 250×4.1 mm i.d.) (Reno, Nevada, USA) cationic exchange column. The chromatographic system was then coupled to the ICP-MS instrument by 20 cm of polytetrafluoroethylene capillary tubing (0.5 mm i.d.) running from the column outlet to the Meinhard nebuliser (obtained from CPI International, Amsterdam, Holland) inlet. For molecular weight fractionation, 10 kDa cut-off filters (Millipore, Bedford, MA, USA) and an Eppendorf (Hamburg, Germany) centrifuge 5804, F34-6-38 were used. Millipore nylon filters (0.45 µm) were used to filter all the HPLC solutions. Reagents and standards Seleno amino acids (SeCys2 and SeMet) were purchased from Sigma Chemical (St. Louis, MO, USA) and dissolved in 3% HCl and deionised Milli-Q water, respectively; inorganic selenium solutions were obtained by dissolving sodium selenite and sodium selenate (Merck, Darmstadt, Germany) in deionised Milli-Q water (Millipore); trimethyselenonium chloride was synthesised in our laboratory following the procedure of Palmer et al. [39]. Stock solutions of 10 mg L–1 were stored in the dark at 4°C and working standard solutions were prepared daily by dilution. A 0.5% (w/v) sodium tetrahydroborate (III) solution was prepared by dissolving NaBH4 powder (Merck, Steinheim, Germany) in deionised Milli-Q water, stabilising in 0.15% (w/v) NaOH and filtering to eliminate turbidity. Hydrochloric acid solution (4 mol L–1) was prepared by diluting concentrated HCl (Merck, suprapur). The 4 mmol L–1 pyridine formate (in 3% methanol) solution, used as eluent for the separation, was prepared by diluting commercial pyridine (Merck) with water and adjusting the pH to 2.8 with formic acid (Merck). HPLC-grade methanol was purchased from Scharlab (Barcelona, Spain). All the HPLC solutions were filtered and degassed before use. H2O2 (35%) from Panreac and HNO3 (65%) pro analysis grade (distilled in our laboratory in a distillation system for acids, model BSB 939IR, Berghof, Germany) were used to digest the samples. The oyster sample analysed was lyophilised material labelled T38 collected in Arcadon (provided by the IRMM centre in Ispra), which was stored at –18 °C following the protocol of CT98-2232 Mulspot Project For the enzymatic hydrolysis of the samples, the non-specific protease subtilisin (Bacillus subtilisin) purchased from Sigma was used. The buffer solution used for enzymatic hydrolysis was 0.1 M Tris (trishydroxymethylaminomethane), obtained from Fluka (Neu Ulm, Germany) at pH=7.5.
Table 1 Operating conditions for Se determination by HPLCICP-MS Forward power Reflected power Coolant Ar flow rate Auxiliary Ar flow rate Nebulisation Ar flow rate Nebuliser type Spray chamber temperature Acquisition mode Integration time Points for peak Flow rate Injection volume Analytical column Mobile phase
1350 W 2.2 W 14.0 L min–1 0.9 L min–1 1.0 L min–1 Meinhard concentric glass 8 °C SIM 2.5 s 399 1.0 mL min–1 100 µL Hamilton PRP X 200 4.0 mmol L–1 pyridine formate (3% methanol), pH=2.8
468 Procedures Sample treatment The T38 sample was prepared by IRMM centre in Ispra with the procedure given, as follows: after the manual de-shelling of the fresh oyster, the wet tissue was minced using a commercial meatmincing instrument, homogenised by means of a special blender (Büchi mixer B-400) and then freeze-dried. For selenium speciation, 250 mg of oyster tissue was hydrolysed following a previously developed method [14]. The procedure consisted of leaching the water soluble selenium and enzymatically hydrolysing both the soluble and non-soluble fractions, which were later ultrafiltered through 10 kDa cut-off filters. The
Table 2 Microwave programme used for digestion of oyster samples Step
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1 2 3 4*
5 15 30 15
15 30 60 30
43 43 43 43
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step requires the addition of H2O2
Table 3 Operating conditions for total Se determination by HG-AAS Intensity of Se EDL lamp Wavelength Slit HCl concentration NaBH4 concentration Sample flow rate Acid flow rate NaBH4 flow rate Ar flow rate
16 mA 196.06 nm 2.0 4 mol L–1 0.5% (w/v) in 0.15% (w/v) NaOH 1.5 mL min–1 1.5 mL min–1 1.5 mL min–1 540 mL min–1
Fig. 1 Scheme of the analytical procedures followed, including the design of the stability studies both for the lyophilised sample and the enzymatic extracts
enzymatic hydrolysis was performed in two steps by adding 10% (w/w) of the non-specific protease subtilisin and incubation at 37 °C and pH 7.5 for 24 h each time. Selenium species were detected by HPLC-ICP-MS using the operating conditions given in Table 1. The calibration was carried out by the standard addition method at m/z 78 and 82, monitoring separately the two isotopes using single ion monitoring (SIM). The negligible differences between the selenium species concentrations, quantified at the two isotopes, leads us to confirm the absence of interferences at the same retention time of the eluted Se species. The spectral interferences from argon species are more significant for the 78 isotope. When the HPLC is coupled to ICP-MS, the argon signal affects in the same way both selenium species and background. The selenium signal was quantified as peak area to base line, correcting this interference. For total selenium determination, the lyophilised sample (250 mg), or the ultrafiltered fractions obtained from the enzymatic hydrolysis, were digested with 2.5 mL of nitric acid and 1 mL of hydrogen peroxide in a microwave oven, following the steps indicated in Table 2. Total selenium concentrations were determined by external and standard addition calibrations (for the lyophilised sample and the hydrolysed extracts, respectively) of the signal obtained from the continuous selenium hydride system connected to AAS equipment in the operating conditions given in Table 3. Se (VI) was reduced to Se (IV) by adding 6 mL of concentrated HCl (of final concentration 6 mol L–1) to the digest and heating at 95 °C for 1 h. The solutions were then diluted to 25 mL with Milli-Q water. A scheme of the different procedures performed is shown in Fig. 1. Design of the stability study The storage containers used for the stability tests on the enzymatic extracts and the freeze-dried sample were 25 mL vials (2×8 cm) and 250 mL bottles (5×12 cm), respectively made of polyethylene and Pyrex. Vials and bottles were previously washed and immersed in a 10% HNO3 bath for 24 h and rinsed with Milli-Q water several times before use. For the stability test performed on the freeze-dried oyster, different portions of solid (previously homogenised by 10 min of manual stirring) were placed in 250 mL polyethylene and Pyrex containers, which were sealed with parafilm and maintained in the
469 Table 4 Reference concentrations (time=0) and standard deviation values obtained for total selenium and the selenium species used in the stability studies Fraction
Total seleTMSe+ nium (µg g–1) (µg g–1)
Lyophilised oyster(1) Water-soluble fraction(2) Non-soluble fraction(2) Soluble+ non-soluble(2)
1.22±0.03(1) 0.40±0.03(3)
0.056±0.009(4) 0.21±0.07(4)
0.50±0.05(3)
0.056±0.013(4) 0.35±0.04(4)
0.90±0.06(3)
0.11±0.02(4)
SeMet (µg g–1)
0.56±0.08(4)
dark at –18, 4 and 20 °C. The reference concentrations (Cref) for the total Se and the Se species identified in the sample were evaluated by ten independent analyses before beginning the stability test. Measurements for the stability study were performed after 1, 7, 15, 30, 90, 180 and 365 days, preparing the sample for total Se and Se speciation immediately before the analysis. The stability test on the enzymatic extracts was performed as follows: 40 enzymatic extracts, equally treated, were mixed and placed into 20 polyethylene and 20 Pyrex vials of 25 mL and sealed with parafilm. Ten vials of each container were maintained in the dark at –18 and 4 °C. A total of ten vials were prepared for storage under each experimental condition and each vial was just used once. After the samples were prepared and stored under the different conditions, three extracts of each type were analysed af-
1Microwave
digestion of the lyophilised oyster and later determination by HG-AAS 2Aqueous extraction, enzymatic hydrolysis and ultrafiltration using 10 kDa cut-off filters 3Microwave digestion of the extracts and later total Se determination by HG-AAS 4Se speciation in the extracts by HPLC-ICP-MS
Fig. 2a–c Chromatograms of a 10 ppb of Se as Se species obtained for cationic exchange chromatography (using 4 mM pyridine, pH=2.8, 1 ml/min); b the liquid extract after enzymatic hydrolysis and ultrafiltration with 10 kDa cut-off filters obtained at reference time; c the solid residue after enzymatic hydrolysis and ultrafiltration with 10 kDa cut-off filters obtained at reference time
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470 ter 1, 7 and 15 days, respectively. The reference concentration values (Cref) were obtained from three different vials by analysis after the sample preparation.
Results and discussion The stability Rx, expressed as a percentage, was evaluated as the ratio of the average of three concentration measurements made under each storage condition (Cx) to the mean
Fig. 3 Total Se stability in the lyophilised oyster stored at 20, 4 and –18 °C in Pyrex and polyethylene containers: dots stability Rx=[(Cx)/(Cref)]×100; Ι uncertainty for each point Ux=(CVx2+ CVref2)1/2×Rx; dashes uncertainty associated with t=0
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The uncertainty Ux in the ratio Rx was obtained from the coefficient of variation of each set of measurements according to the following expression [40]: 1/2 Ux = CV2x +CV2ref ×Rx (2) Where CVx is the coefficient of variation of three independent measurements under each storage condition and CVref is the coefficient of variation obtained for the reference conditions. In the case of ideal stability, Rx should be 100%, but in practice there are random variations due to the uncertainty
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in the measurements because of the standard deviation observed, therefore, the value Rx should be between the limits [100-Ux] and [100+Ux] to conclude the species stability [40]. Stability of freeze-dried oyster The stability of total selenium and selenium species was determined in an oyster candidate reference material labelled T38, previously used for an intercomparison exercise. The freeze-dried sample, stored at –18 °C in dark Pyrex bottles, was analysed immediately after it was received. The analytical method used, its analytical characteristics
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and the results obtained for total Se and Se species have been reported in a previous paper [14]. Table 4 shows the reference concentration and the standard deviation values obtained for total selenium and selenium species, which were used in the stability tests. Figure 2 shows the chromatogram obtained for (a) 10 ppb of Se as Se species obtained for cationic exchange chromatography (using 4 mM pyridine, pH=2.8, 1 mL/min) and the initial chromatograms obtained for the hydrolysed oyster (b) soluble fraction, and (c) non-soluble fraction under the same chromatographic conditions. The results obtained for total selenium after 1, 7, 15, 30, 90, 180 and 365 days are shown in Fig. 3, and demonstrate that total Se in the freeze-dried material is stable and does not depend either on the container or on the storage temperature Figures 4 and 5 show the results obtained for the selenium species (SeMet and TMSe+, respectively) in stability
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Fig. 5 TMSe+ species stability in lyophilised oyster stored at 20, 4 and –18 °C in Pyrex and polyethylene containers
tests after 1, 7, 15, 30, 90, 180 and 365 days of the same storage conditions as in the total selenium studies. These results allow us to conclude that the identified selenium species are stable under all the storage condition tested, for at least one year. Therefore, the distribution and storage of the lyophilised oyster material could be carried out without any special considerations as to container and temperature. Stability tests on enzymatic extracts The total Se stability tests on the enzymatic extracts were carried out in the water-soluble and water-insoluble fractions separately, to check for a possible relationship between the sample instability and matrix complexity.
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The storage conditions were similar to those chosen in the above study, except that 20 °C was excluded in order to avoid the microbiological activity which may cause sample decomposition. The results obtained (Fig. 6) show that total selenium in the soluble fraction after enzymatic hydrolysis and storage at 4 °C in polyethylene or Pyrex containers, was stable for at least 30 days. However, at –18 °C, significant losses were detected within 7 days in both containers. The total Se content in the non-soluble fraction was stable for about 15 days in the best storage conditions (4 °C and Pyrex containers) while significant losses were detected in 1 day in polyethylene vials at 4 and –18 °C and in Pyrex at –18 °C. Some reasons for losses at –18 °C are related to the possible higher transformation rate and loss of species during the storage at –18 °C. These are due to the freezing and melting processes where the solid and liquid phases coexist, which is in agreement with Lindemann et al. [17] and Feldmann et al. [41].
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The differences found between the soluble and nonsoluble fractions could be due to the high complexity of the non-soluble fraction and to adsorption of higher molecular weight proteins to the container walls. Figure 7 shows the stability results obtained for the SeMet, as the sum of the species found in both fractions (soluble and non-soluble). From these results, no instability (R±U) could be attributed to SeMet species for 10 days under the storage conditions tested (–18 and 4 °C, and polyethylene and Pyrex containers). However, almost to-
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tal losses were observed within 15 days (the concentration found was lower than the quantification limit). The instability of SeMet after 10 days should affect the total selenium stability, although total selenium in the extracts is stable for a longer time (15 days). This can be explained as a possible transformation of this species to a new one that may be highly retained in the chromatographic column, as any new peak is observed in the chromatogram obtained after 15 days of storage (Fig. 8). On the other hand, TMSe+ (Fig. 9) was stable for at least 15 days under the conditions tested (–18 and 4 °C, and polyethylene and Pyrex containers).
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Fig. 7 SeMet species stability in the enzymatic extracts, expressed as the sum of the concentrations obtained for soluble and non-soluble fractions
Fig. 8a,b Chromatograms of a the liquid extract and b the solid residue, both after enzymatic hydrolysis and ultrafiltration with 10 kDa cut-off filters obtained after 15 days of storage at 4 °C in Pyrex containers
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Conclusions The stability studies performed on lyophilised oysters offer important information for ensuring an adequate transport and storage conditions without losses in total selenium and selenium species concentrations. Total selenium and the Se species TMSe+ and SeMet in the freeze-dried sample T38 were stable for at least one year at –18, 4 and 20 °C stored in either polyethylene or Pyrex containers. Therefore, the transport and storage of this freeze-dried material can be carried out at room temperature using polyethylene containers. The best storage conditions for the enzymatic extracts of the soluble fraction were 4 °C in both polyethylene and Pyrex containers in terms of total Se, as no significant losses were observed for at least 30 days. However, the enzymatic extracts of the non-soluble fraction was stable for 30 days at 4 °C in Pyrex containers. SeMet was stable for 10 days in the enzymatic extracts at +4 and –18 °C in both Pyrex and polyethylene containers, and TMSe+ was stable under these conditions for at least 15 days. From these results, it can be concluded that the enzymatic extracts can be stored for a maximum of 10 days before analysis, the best conditions being at 4 °C in Pyrex containers. Acknowledgements The authors are grateful for financial support from the Measurements and Testing Programme (EC) under Project CT98-2232 and DGICYT n° PB98-076.
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