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A selective optosensor for UV spectrophotometric determination of thiamine in the presence of other vitamins B
Analytica Chimica Acta 376 (1998) 227±233
A selective optosensor for UV spectrophotometric determination of thiamine in the presence of other vitamins B P. Ortega Barrales, M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz* Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of JaeÂn, Paraje Las Lagunillas, s/n, E-23071 JaeÂn, Spain Received 6 May 1998; received in revised form 22 July 1998; accepted 31 July 1998
Abstract A ¯ow-through UV-photometric sensor for the rapid, sensitive and selective determination of thiamine in the presence of ribo¯avin, pyridoxine and hydroxocobalamine was developed. The active solid phase of the optosensor was the cationic resin Sephadex CMC-25 placed in a quartz ¯ow cell of 1 mm of optical path length. Thiamine was transitorily retained on this active microzone and its intrinsic absorbance monitored at 247 nm. Three calibration lines were constructed by using 300, 600, and 1000 ml, respectively of injected sample volume. Their linear dynamic ranges were 2.0±33.0, 1.0±20.0 and 0.6± 12.0 mg mlÿ1 and their RSD (%) 0.8, 0.9 and 1.8 for the determination of 28, 15 and 9 mg mlÿ1, respectively, with sampling frequency of 18, 16 and 14 hÿ1. The chemicals of the B-complex commonly found along with thiamine were tolerated at w/w ratios higher than 10, so they did not cause interference at the usual concentrations. The sensor was successfully applied to the determination of thiamine in pharmaceuticals containing only thiamine or other B vitamins as well. # 1998 Published by Elsevier Science B.V. Keywords: Thiamine; Sephadex CMC-25; Solid phase spectrometry; Optosensor; Pharmaceutical preparations
1. Introduction Solid phase spectrometry (SPS), i.e. solid phase preconcentration used in combination with direct optical non-destructive spectrometry in the solid phase itself consists of solid phase spectrophotometry (SPSP) and solid phase spectro¯uorimetry (SPSF). This is a relatively recent technique which has demonstrated its applicability to a wide variety of real samples [1±6]. It shows several intrinsic features such as a strong increase in selectivity, lower detection *Corresponding author. Tel.: +34-53-21-21-47; fax: +34-53-2121-41. 0003-2670/98/$19.00 # 1998 Published by Elsevier Science B.V. PII S0003-2670(98)00540-6
limits and a very high increase in sensitivity as compared to the same procedures in homogeneous solution spectrophotometry or spectro¯uorimetry. However, SPSP in the UV region has been scarcely exploited. SPS integrated with ¯ow injection analysis (FIA) is also a new technique based on the retention of the analyte (or any of the components of a chemical reaction) on an active solid support placed in an appropriate ¯ow cell by using a non-destructive optical detector (photometric or ¯uorimetric) [7±10]. The retained species are eluted after reaching the detection zone and developing the analytical signal, in order to keep the active support ready for the next sample, i.e.
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to make it reusable. These SPS±FIA systems are named ¯ow-through optical sensors [11±14] or ¯ow-through optical sensing devices [15]. Over the last few years, an increasing interest has been payed to these systems, and a need for further efforts to apply them to real analytical problems (for example, pharmaceutical samples) [16] has been felt. One of the most interesting cases of these optical sensors occurs when the carrier itself elutes the species retained, thus originating a transient retention of it and, consequently, a transient signal without showing alterations in the baseline due to swelling and unswelling when using an ion exchange resin as a solid support (as usual) that should reduce the sampling rate and the resin's lifetime [1]. In a previous paper [17], we proposed a very sensitive and selective (batch mode) solid phase UV spectrophotometric procedure for microdetermination of vitamin B1 in the presence of vitamins B2, B6 and B12. Nevertheless, it is rather slow and needs relatively high volumes of sample solution. With the aim of improving the features of that method, in this paper an integrated SPSP±FIA method (i.e. optical ¯owthrough sensor) for determination of thiamine is developed, based on the transient retention of the analyte on Sephadex CMC-25 cation exchanger in the ¯ow cell and detection of its intrinsic absorbance on the UV region at 247 nm. Compared with the previous batch procedure, this FIA method offers considerable economy with regard to sample, reagent and solid support consumption, and the time required for the analysis without any loss of precision. It is a reusable sensor inasmuch as three steps (retention, elution and detection) take place simultaneously in each determination. These features make it very suitable for routine analyses in control laboratories, as shown in this work by applying it to real samples without any pretreatment of the sample and with a very good sample throughput. 2. Experimental 2.1. Reagents Unless otherwise stated, all solutions were prepared from analytical-reagent grade chemicals by using doubly distilled water.
Thiamine stock solution, 1000 mg lÿ1 (as the hydrochloride), was prepared from thiamine hydrochloride (Fluka). This solution was stable for at least one month in a refrigerator at 4±58C. Work solutions were prepared fresh daily by dilution to the appropriate volume with water. Sephadex CMC-25 (Aldrich) cation exchanger (40± 120 mm; capacity: 4.3 meq gÿ1) was used in the H form without any pretreatment, and packed in a ¯owthrough cell for measurement of solid phase UV light absorption, with glass wool placed at the bottom of the cell to keep the resin beads free from the possible movements caused by the carrier. A sodium acetate/acetic acid buffer NaAc/HAc solution 0.15 M (pH 4.8) was used as carrier solution, itself being the eluent solution. 2.2. Instrumentation A Unicam 8625 UV/V microprocessor controlled spectrophotometer (from UNICAM Analytical Systems) was used, equipped with a Hellma 138-QS ¯ow cell (1 mm optical path length and 40 ml inner volume). It was connected to an IBM Personal System/2 Model 80286 computer running the 8625 Series Rate Software, and to an EPSON EPL-5200 printer. A four-channel Gilson Minipuls-3 peristaltic pump with rate selector, a Rheodyne Model 5041 injection valve and te¯on tubing of 0.8 mm i.d. were also used. 2.3. Manifold and procedure The ¯ow-injection manifold used is shown in Fig. 1. The aqueous solution containing thiamine is injected by the injection valve into the carrier solution (buffer NaAc/HAc pH 4.8) ¯owing at 0.74 ml minÿ1. The injected volume depends on the concentration of thiamine: 300, 600 and 1000 ml for concentrations ranging from 2.0 to 33.0, from 1.0 to 20.0, or from 0.6 to 12.0 mg mlÿ1, respectively. The plug is transported to the ¯ow cell containing the packed resin, the transitory thiamine retention/detection at 247 nm taking place, and the carrier itself then elutes and regenerates the active microzone of the sensor. When the analytical signal comes back to its baseline, the next sample is injected and determined as before.
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229
Fig. 1. Schematic diagram of the FIA system: C/E: carrier/eluent; PP: peristaltic pump; V: injection valve; S: sample; SP: spectrophotometric detector; FC: 1 mm quartz flow cell with active microzone of the sensor; R: Sephadex CMC-25; CM: computer; P: printer; W: waste.
Use the peak height (maximum absorbance) as a measure of absorbance throughout for the samples and calibration runs. 3. Results and discussion 3.1. Preliminary study Spectral features of the analyte in homogeneous solution and on solid sorbent were previously established in batch mode [17]. Several Sephadex type solid supports were tested here for the FIA sensor (SPC-25, CMC-25, CMC-50, G-15, G-75 and G-100) and the best results were found by using the Sephadex CMC-25 cation exchanger. Dowex resins were not considered for the sorption test due to their high absorption in the UV region. The absorption maximum of thiamine sorbed on the active microzone of the sensor appears at 247 nm, thus showing a very slight bathochromic shift with respect to the homogeneous solution (246 nm). The recording rate used in this preliminary study was 250 nm minÿ1, and from these spectra the wavelength chosen for all measurements was 247 nm. At this wavelength, the solid support absorbance measured against air was about 1.100.
As a result of the preconcentration process of thiamine on the active sensing microzone, FIA recordings obtained with the ¯ow cell of 1 mm optical path packing with Sephadex CMC-25 showed a dramatic increase in the absorbance with respect to the use of the ¯ow cell without packing with solid sorbent: about 11-times higher than that obtained in the latter case. 3.2. Optimization of the variables The chemical and FIA variables involved in the system as well as those of the packing of the solid support in the ¯ow cell (retention/detection unit variable) were optimized. 3.2.1. Level of the solid sorbent in the flow cell The level of the support in the ¯ow cell is a very important variable; if it falls below the bottom of the light beam, the measurement of the absorbance of the analyte is carried out only in solution (Zone I in Fig. 2(a)) and absorbance remains constant. As the level of the resin increases and this is irradiated by the light beam, an increase in absorbance is observed as in this case the measurement is partially performed on the resin (Zone II in Fig. 2(a)). Finally, when the level
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Fig. 2. Influence of some variables on the analytical response and elution time: (a) level of resin in the flow cell, I, III: dark zones, II: flow cell irradiated area; (b) concentration of carrier/eluent; (c) flow rate.
of the resin reaches the top of the light beam, the measurement is completely performed on the resin (Zone III in Fig. 2(a)). If the level of the resin is increased in this zone, the highest analyte concentration is ®xed outside the detection area and absorbance decreases. The optimum height of the solid support in the ¯ow cell was 15 mm from the bottom of the cell (see Fig. 2(a)). 3.2.2. Optimization of chemical variables Several buffering solutions of various concentrations were tested as carrier/eluent solutions: citric acid±sodium hydroxide (pH 2.5±4.0), acetic acid± sodium acetate (pH 3.5±5.5), hexamethylenetetramine±hydrochloride acid (pH 4.0±6.0), and sodium dihydrogen phosphate±sodium hydroxide (pH 5.0± 7.0). The results showed that the acetate buffer of pH 4.8 was the most suitable solution because it produced a higher analytical signal and reduced the time for the complete return to base line, at the same time increasing the sampling frequency. In this way, the carrier solution being the eluent, no swelling and unswelling of the resin are produced and a more stable baseline and a higher sampling frequency are obtained with respect to the case in which the eluent is different from the carrier solution [8]. When the concentration of carrier/eluent chosen (pH 4.8) was varied, the effect showed in Fig. 2(b) was observed. As a compromise between a higher analytical signal and a lower eluting time (higher sampling frequency), a buffer concentra-
tion of 0.15 M was chosen. In this way, a dramatic decrease in eluting time (40%) was obtained with only a light decrease in absorbance (about 6%) with respect to using a 0.1 M concentration. 3.2.3. Optimization of FI variables The absorbance increased linearly (A0.074 7.9110ÿ4v, r0.9984) with increasing injection volume (v), from 100 to 1500 ml (Fig. 3(a)). This is due to the transient ®xation of a higher amount of analyte on the sensing microzone as the injected volume increases, and it allows: to work with a wide range of analyte concentrations, after calibration of the sensor, by simply changing the sample volume injected, and/or to reduce matrix effects by appropriate dilution of the samples. This effect is similar to that found in SPS methodology [1,2]. The ¯ow rate was changed from 0.42 to 1.47 ml minÿ1 using 600 ml of 2.9510ÿ5 M thiamine hydrochloride solution. The analytical signal and the elution time decreased (Fig. 2(c)). As a compromise, a ¯ow rate of 0.74 ml minÿ1 was chosen. Its signal decrease was 13% against 32% decrease of elution time in comparison, with the signal and elution time for 0.42 ml minÿ1, respectively. Because of the absence of derivatizing reaction, the length of the transport system between the injection valve and the ¯ow cell was the minimum allowing both units to be connected. It minimizes the dispersion and allows a higher sampling frequency.
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Fig. 3. (a) Influence of the injection volume on the analytical response; 2.9510ÿ5 mol lÿ1 thiamine. (b) Calibration lines for 300, 600 and 1000 ml of injected volume.
3.3. Analytical features of the proposed method In the working conditions above established the calibration lines obtained according to the procedure described for the three injection volumes are shown in Fig. 3(b). The sensitivity is enhanced by increasing the injection volume. This increase can be calculated from the slope ratios (S) of the calibration graphs for the three volumes used in this study: S1000/3002.44; S600/300 1.74 and S1000/6001.44 (the subscripts represent the injection volume, ml). The sensitivity of the method proposed has been compared with that of the conventional FIA method (solution): for a 600 ml injection volume the sensitivity is more than 11 times higher than that of the corresponding solution method. The
sampling frequency decreased lightly as the injection volume increased. Table 1 summarizes the analytical ®gures of merit. The advantages obtained in sensitivity by increasing the injected sample volume are self-evident. 3.4. Effect of foreign species The effect of foreign species on the determination of 11 mg mlÿ1 of thiamine by the proposed procedure was carried out for 600 ml of sample volume. The species tested include other vitamins of the B complex and those commonly present along with thiamine. This study was undertaken by adding a known amount of foreign species to the thiamine hydrochloride solution of 11 mg mlÿ1. The highest concentration of
Table 1 Analytical features of the method Injected volume (ml)
Linear dynamic range (mg mlÿ1)
Equation (r)
RSD (%)
Detection limit (mg mlÿ1)
Sampling frequency (hÿ1)
300 600 1000
2.0±33.0 1.0±20.0 0.6±12.0
A0.0000.031c (r0.9999) A0.0170.054c (r0.9995) A0.0160.078c (r0.9994)
0.75a 0.89b 1.77c
0.42 0.25 0.16
18 16 14
Aabsorbance; cconcentration of thiamine; rregression coefficient. 28 mg mlÿ1 of thiamine. b 15 mg mlÿ1 of thiamine. c 9 mg mlÿ1 of thiamine. a
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Table 2 Effect of foreign species on the determination of 11 mg mlÿ1 of thiamine Foreign species
Tolerance mg mlÿ1 interferent/mg mlÿ1 thiamine
L-Carnitine, L-Lysine
>100 50 >40 20 18 16 10
Nicotinic acid, folic acid Biotine Ascorbic acid Vitamin B12 Vitamin B6 Nicotinamide, vitamin B2
foreign species tested was 1000 mg mlÿ1, except for biotine which was 440 mg mlÿ1. A foreign species is considered not to interfere if it produces an error not exceeding 5% in the determination of the analyte. Table 2 shows the results obtained. The method proposed shows a high tolerance level to all the species tested, which are frequently encoun-
tered along with thiamine including other vitamins and B-complex factors. The tolerance level to all these species is higher than the concentration usually found for them in pharmaceuticals containing thiamine. Moreover, the high tolerance level to the other B group vitamins (namely B12, B6 and B2) proves the high selectivity shown by the method for thiamine determination in the presence of these vitamins by using a spectrophotometric detector tunned in UV region. On the other hand, if the tolerance level of the method proposed to vitamins of the B group is compared with that found by conventional UV spectrophotometry at the same pH [17], we ®nd that the amount tolerated in the method developed in this paper is 72, 75 and 770 times higher than that shown by UV conventional spectrophotometry in homogeneous solution. Due to its cationic nature, vitamin B1 is selectively ®xed on the Sephadex CMC-25 cation exchanger (sensing microzone), and it strongly raises
Table 3 Determination of thiamine in pharmaceutical preparations Pharmaceutical preparation
b
Antineurine Astenolitc Hepacoban B12d Neurodavure Nervobionf Benervag Salvacolonh Trimetaboli a
Stated thiamine HCl content (mg)
125 50 (thiamine NOÿ 3) 50 250 100 (thiamine NOÿ 3) 300 48 600
Thiamine HCl founda (mgRSD (%)) Proposed method
Fluorimetric method
128.6 (0.9) 51.6 (1.7) 49 (2) 251.7 (0.6) 102 (1) 302.3 (0.8) 46.0 (0.8) 605.3 (0.7)
129.7 (1.8) 50.6 (0.9) 48 (1) 255.7 (0.2) 103 (2) 297.0 (0.7) 48 (2) 589.0 (0.5)
Data are based on the average obtained from three determinations. Injectable vials (from Tedec. Meiji. Farma., Madrid, Spain) containing thiamine 125 mg, pyridoxine 125 mg, cyanocobalamin 5000 mg, and didocaine 3 mg. c Drinking ampoules (from Elmu QuõÂmica FCA, Madrid, Spain) containing thiamine 50 mg, pyridoxine 50 mg, cyanocobalamin 50 mg, acetylglutamine 50 mg, inositol 30 mg, acetylaspartic acid 70 mg, carnitine 1000 mg, potassium aspartate 75 mg, magnesium aspartate 75 mg, ethanol 96% 400 mg, and excip. sodium saccharin 40 mg. d Injectable vials (from Bohm, Madrid, Spain) containing thiamine 50 mg, cyanocobalamin 30 mg, hepatic extract (B12 native) 60 mg, and calcium pantothenate 5 mg. e Capsules (from Belmac, Madrid, Spain) containing thiamine 250 mg, pyridoxine 250 mg, hydroxocobalamine acetate 25 mg, and excip. magnesium stearate csp 1 capsule. f Capsules (from E. Merck-Darmstadt Igoda, Barcelona, Spain) containing thiamine 100 mg, cyanocobalamin 1000 mg, and pyridoxine 100 mg. g Tablets (from Roche Nicholas, Barcelona, Spain) containing thiamine 300 mg. h Solid vial (from SALVAT, Barcelona, Spain) containing thiamine 48 mg, cyanocobalamin 144 mg, riboflavin 12 mg, pyridoxine 120 mg, calcium pantothenate 240 mg, nicotinamide 480 mg, lysine 7200 mg, excip. sodium saccharin 59.8 mg, and sucrose 6080 mg. i Syrup (from Uriach-Biohorm, Barcelona, Spain) containing in 100 ml thiamine 600 mg, pyridoxine 600 mg, cyanocobalamin 20 mg, methopine 35 mg, lysine hydrochloride 5 g, carnitine hydrochloride 75 mg, sorbitol 70% 20 g, and sucrose 45 g. b
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the tolerance level to the presence of the B vitamins (and of other B complex factors). 3.5. Analytical applications Pharmaceutical preparations containing thiamine hydrochloride, either alone or in mixtures with other vitamins of the B group or other ingredients of a different nature, were assayed by the method proposed for an injection volume of 600 ml; for the sake of comparison, the thiamine content of these pharmaceuticals was also determined by the AOAC ¯uorimetric method [18] (based on the oxidation of thiamine to thiochrome by potassium ferrocyanide) used as a reference method. In every pharmaceutical preparations the results were found to be very good (Table 3), thus con®rming the validity of the sensor proposed in this paper. 4. Conclusions The continuous ¯ow-through UV spectrophotometric sensor here developed for determination of thiamine, based on the transient retention of the analyte in the ¯ow cell and monitorization of its intrinsic UV absorbance, shows clear advantages with respect to the conventional UV spectrophotometric method, namely a very important improvement in sensitivity, selectivity, simplicity, and speed. These are achieved because the cation exchange nature of the solid support placed in the cell preconcentrates and selectively ®xes the cation thiamine in the detection region (active sensing microzone), thus preventing those non-cationic species present along with thiamine to interfere. Moreover, these features in combination with an FIA system in which the carrier itself regenerates the active microzone, make the sensor a cheap, continuous, simple, and reusable sensing device suitable for determination of thiamine in pharmaceuticals. If compared to the solid phase UV spec-
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trophotometric batch method previously developed [17], the present method is much faster and cheaper (because the sample, buffer solution and solid active support consumption are much smaller), much less tedious (for example, no weight of a known amount of resin is necessary for each determination), and more precise, although less sensitive due to the use of a much smaller volume of sample (up to 3000 times less). References [1] P. Ortega Barrales, M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz, Anal. Chim. Acta 360 (1998) 143. [2] M.L. FernaÂndez de CoÂrdova, A. Ruiz Medina, A. Molina DõÂaz, Fresenius' J. Anal. Chem. 357 (1997) 44. [3] L.F. CapitaÂn-Vallvey, M.D. FernaÂndez, I. De Orbe, R. Avidad, Analyst 120 (1995) 2421. [4] F. CapitaÂn, E.J. Alonso, R. Avidad, L.F. CapitaÂn-Vallvey, J.L. Vilchez, Anal. Chem. 65 (1993) 1336. [5] L.F. CapitaÂn-Vallvey, N. Navas Iglesias, I. De Orbe PayaÂ, R. Avidad, Talanta 43 (1996) 1457. [6] M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz, M.I. Pascual Reguera, L.F. CapitaÂn-Vallvey, Talanta 42 (1995) 1057. [7] K. Yoshimura, Anal. Chem. 59 (1987) 2922. [8] L.F. CapitaÂn-Vallvey, M.C. Valencia, G. MiroÂn, Anal. Chim. Acta 289 (1994) 365. [9] P. Richter, M.D. Luque de Castro, M. ValcaÂrcel, Anal. Lett. 25 (1992) 2279. [10] K. Yoshimura, S. Matsuoka, T. Tabuchi, H. Waki, Analyst 117 (1992) 189. [11] M. ValcaÂrcel, M.D. Luque de Castro, Flow-through (Bio)chemical Sensors, Chapter 2, Elsevier, Amsterdam, 1994. [12] R.M. Lin, D.J. Lin, A.L. Sun, Talanta 40 (1993) 381. [13] Z. Gong, Z. Zhang, Anal. Chim. Acta 339 (1997) 161. [14] Y. Wang, K. Wang, W. Liu, G. Shen, R. Yu, Analyst 122 (1997) 69. [15] D. Chen, M.D. Luque de Castro, M. ValcaÂrcel, Anal. Chim. Acta 261 (1992) 269. [16] M. ValcaÂrcel, M.D. Luque de Castro, Analyst 118 (1993) 593. [17] P. Ortega Barrales, M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz, Anal. Chem. 70 (1998) 271. [18] AOAC Official Methods of Analysis, vol. 2, 15th ed., Association of Official Analytical Chemists, Inc, Arlington, VA, 1990, p. 1050.
A selective optosensor for UV spectrophotometric determination of thiamine in the presence of other vitamins B P. Ortega Barrales, M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz* Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of JaeÂn, Paraje Las Lagunillas, s/n, E-23071 JaeÂn, Spain Received 6 May 1998; received in revised form 22 July 1998; accepted 31 July 1998
Abstract A ¯ow-through UV-photometric sensor for the rapid, sensitive and selective determination of thiamine in the presence of ribo¯avin, pyridoxine and hydroxocobalamine was developed. The active solid phase of the optosensor was the cationic resin Sephadex CMC-25 placed in a quartz ¯ow cell of 1 mm of optical path length. Thiamine was transitorily retained on this active microzone and its intrinsic absorbance monitored at 247 nm. Three calibration lines were constructed by using 300, 600, and 1000 ml, respectively of injected sample volume. Their linear dynamic ranges were 2.0±33.0, 1.0±20.0 and 0.6± 12.0 mg mlÿ1 and their RSD (%) 0.8, 0.9 and 1.8 for the determination of 28, 15 and 9 mg mlÿ1, respectively, with sampling frequency of 18, 16 and 14 hÿ1. The chemicals of the B-complex commonly found along with thiamine were tolerated at w/w ratios higher than 10, so they did not cause interference at the usual concentrations. The sensor was successfully applied to the determination of thiamine in pharmaceuticals containing only thiamine or other B vitamins as well. # 1998 Published by Elsevier Science B.V. Keywords: Thiamine; Sephadex CMC-25; Solid phase spectrometry; Optosensor; Pharmaceutical preparations
1. Introduction Solid phase spectrometry (SPS), i.e. solid phase preconcentration used in combination with direct optical non-destructive spectrometry in the solid phase itself consists of solid phase spectrophotometry (SPSP) and solid phase spectro¯uorimetry (SPSF). This is a relatively recent technique which has demonstrated its applicability to a wide variety of real samples [1±6]. It shows several intrinsic features such as a strong increase in selectivity, lower detection *Corresponding author. Tel.: +34-53-21-21-47; fax: +34-53-2121-41. 0003-2670/98/$19.00 # 1998 Published by Elsevier Science B.V. PII S0003-2670(98)00540-6
limits and a very high increase in sensitivity as compared to the same procedures in homogeneous solution spectrophotometry or spectro¯uorimetry. However, SPSP in the UV region has been scarcely exploited. SPS integrated with ¯ow injection analysis (FIA) is also a new technique based on the retention of the analyte (or any of the components of a chemical reaction) on an active solid support placed in an appropriate ¯ow cell by using a non-destructive optical detector (photometric or ¯uorimetric) [7±10]. The retained species are eluted after reaching the detection zone and developing the analytical signal, in order to keep the active support ready for the next sample, i.e.
228
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to make it reusable. These SPS±FIA systems are named ¯ow-through optical sensors [11±14] or ¯ow-through optical sensing devices [15]. Over the last few years, an increasing interest has been payed to these systems, and a need for further efforts to apply them to real analytical problems (for example, pharmaceutical samples) [16] has been felt. One of the most interesting cases of these optical sensors occurs when the carrier itself elutes the species retained, thus originating a transient retention of it and, consequently, a transient signal without showing alterations in the baseline due to swelling and unswelling when using an ion exchange resin as a solid support (as usual) that should reduce the sampling rate and the resin's lifetime [1]. In a previous paper [17], we proposed a very sensitive and selective (batch mode) solid phase UV spectrophotometric procedure for microdetermination of vitamin B1 in the presence of vitamins B2, B6 and B12. Nevertheless, it is rather slow and needs relatively high volumes of sample solution. With the aim of improving the features of that method, in this paper an integrated SPSP±FIA method (i.e. optical ¯owthrough sensor) for determination of thiamine is developed, based on the transient retention of the analyte on Sephadex CMC-25 cation exchanger in the ¯ow cell and detection of its intrinsic absorbance on the UV region at 247 nm. Compared with the previous batch procedure, this FIA method offers considerable economy with regard to sample, reagent and solid support consumption, and the time required for the analysis without any loss of precision. It is a reusable sensor inasmuch as three steps (retention, elution and detection) take place simultaneously in each determination. These features make it very suitable for routine analyses in control laboratories, as shown in this work by applying it to real samples without any pretreatment of the sample and with a very good sample throughput. 2. Experimental 2.1. Reagents Unless otherwise stated, all solutions were prepared from analytical-reagent grade chemicals by using doubly distilled water.
Thiamine stock solution, 1000 mg lÿ1 (as the hydrochloride), was prepared from thiamine hydrochloride (Fluka). This solution was stable for at least one month in a refrigerator at 4±58C. Work solutions were prepared fresh daily by dilution to the appropriate volume with water. Sephadex CMC-25 (Aldrich) cation exchanger (40± 120 mm; capacity: 4.3 meq gÿ1) was used in the H form without any pretreatment, and packed in a ¯owthrough cell for measurement of solid phase UV light absorption, with glass wool placed at the bottom of the cell to keep the resin beads free from the possible movements caused by the carrier. A sodium acetate/acetic acid buffer NaAc/HAc solution 0.15 M (pH 4.8) was used as carrier solution, itself being the eluent solution. 2.2. Instrumentation A Unicam 8625 UV/V microprocessor controlled spectrophotometer (from UNICAM Analytical Systems) was used, equipped with a Hellma 138-QS ¯ow cell (1 mm optical path length and 40 ml inner volume). It was connected to an IBM Personal System/2 Model 80286 computer running the 8625 Series Rate Software, and to an EPSON EPL-5200 printer. A four-channel Gilson Minipuls-3 peristaltic pump with rate selector, a Rheodyne Model 5041 injection valve and te¯on tubing of 0.8 mm i.d. were also used. 2.3. Manifold and procedure The ¯ow-injection manifold used is shown in Fig. 1. The aqueous solution containing thiamine is injected by the injection valve into the carrier solution (buffer NaAc/HAc pH 4.8) ¯owing at 0.74 ml minÿ1. The injected volume depends on the concentration of thiamine: 300, 600 and 1000 ml for concentrations ranging from 2.0 to 33.0, from 1.0 to 20.0, or from 0.6 to 12.0 mg mlÿ1, respectively. The plug is transported to the ¯ow cell containing the packed resin, the transitory thiamine retention/detection at 247 nm taking place, and the carrier itself then elutes and regenerates the active microzone of the sensor. When the analytical signal comes back to its baseline, the next sample is injected and determined as before.
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Fig. 1. Schematic diagram of the FIA system: C/E: carrier/eluent; PP: peristaltic pump; V: injection valve; S: sample; SP: spectrophotometric detector; FC: 1 mm quartz flow cell with active microzone of the sensor; R: Sephadex CMC-25; CM: computer; P: printer; W: waste.
Use the peak height (maximum absorbance) as a measure of absorbance throughout for the samples and calibration runs. 3. Results and discussion 3.1. Preliminary study Spectral features of the analyte in homogeneous solution and on solid sorbent were previously established in batch mode [17]. Several Sephadex type solid supports were tested here for the FIA sensor (SPC-25, CMC-25, CMC-50, G-15, G-75 and G-100) and the best results were found by using the Sephadex CMC-25 cation exchanger. Dowex resins were not considered for the sorption test due to their high absorption in the UV region. The absorption maximum of thiamine sorbed on the active microzone of the sensor appears at 247 nm, thus showing a very slight bathochromic shift with respect to the homogeneous solution (246 nm). The recording rate used in this preliminary study was 250 nm minÿ1, and from these spectra the wavelength chosen for all measurements was 247 nm. At this wavelength, the solid support absorbance measured against air was about 1.100.
As a result of the preconcentration process of thiamine on the active sensing microzone, FIA recordings obtained with the ¯ow cell of 1 mm optical path packing with Sephadex CMC-25 showed a dramatic increase in the absorbance with respect to the use of the ¯ow cell without packing with solid sorbent: about 11-times higher than that obtained in the latter case. 3.2. Optimization of the variables The chemical and FIA variables involved in the system as well as those of the packing of the solid support in the ¯ow cell (retention/detection unit variable) were optimized. 3.2.1. Level of the solid sorbent in the flow cell The level of the support in the ¯ow cell is a very important variable; if it falls below the bottom of the light beam, the measurement of the absorbance of the analyte is carried out only in solution (Zone I in Fig. 2(a)) and absorbance remains constant. As the level of the resin increases and this is irradiated by the light beam, an increase in absorbance is observed as in this case the measurement is partially performed on the resin (Zone II in Fig. 2(a)). Finally, when the level
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Fig. 2. Influence of some variables on the analytical response and elution time: (a) level of resin in the flow cell, I, III: dark zones, II: flow cell irradiated area; (b) concentration of carrier/eluent; (c) flow rate.
of the resin reaches the top of the light beam, the measurement is completely performed on the resin (Zone III in Fig. 2(a)). If the level of the resin is increased in this zone, the highest analyte concentration is ®xed outside the detection area and absorbance decreases. The optimum height of the solid support in the ¯ow cell was 15 mm from the bottom of the cell (see Fig. 2(a)). 3.2.2. Optimization of chemical variables Several buffering solutions of various concentrations were tested as carrier/eluent solutions: citric acid±sodium hydroxide (pH 2.5±4.0), acetic acid± sodium acetate (pH 3.5±5.5), hexamethylenetetramine±hydrochloride acid (pH 4.0±6.0), and sodium dihydrogen phosphate±sodium hydroxide (pH 5.0± 7.0). The results showed that the acetate buffer of pH 4.8 was the most suitable solution because it produced a higher analytical signal and reduced the time for the complete return to base line, at the same time increasing the sampling frequency. In this way, the carrier solution being the eluent, no swelling and unswelling of the resin are produced and a more stable baseline and a higher sampling frequency are obtained with respect to the case in which the eluent is different from the carrier solution [8]. When the concentration of carrier/eluent chosen (pH 4.8) was varied, the effect showed in Fig. 2(b) was observed. As a compromise between a higher analytical signal and a lower eluting time (higher sampling frequency), a buffer concentra-
tion of 0.15 M was chosen. In this way, a dramatic decrease in eluting time (40%) was obtained with only a light decrease in absorbance (about 6%) with respect to using a 0.1 M concentration. 3.2.3. Optimization of FI variables The absorbance increased linearly (A0.074 7.9110ÿ4v, r0.9984) with increasing injection volume (v), from 100 to 1500 ml (Fig. 3(a)). This is due to the transient ®xation of a higher amount of analyte on the sensing microzone as the injected volume increases, and it allows: to work with a wide range of analyte concentrations, after calibration of the sensor, by simply changing the sample volume injected, and/or to reduce matrix effects by appropriate dilution of the samples. This effect is similar to that found in SPS methodology [1,2]. The ¯ow rate was changed from 0.42 to 1.47 ml minÿ1 using 600 ml of 2.9510ÿ5 M thiamine hydrochloride solution. The analytical signal and the elution time decreased (Fig. 2(c)). As a compromise, a ¯ow rate of 0.74 ml minÿ1 was chosen. Its signal decrease was 13% against 32% decrease of elution time in comparison, with the signal and elution time for 0.42 ml minÿ1, respectively. Because of the absence of derivatizing reaction, the length of the transport system between the injection valve and the ¯ow cell was the minimum allowing both units to be connected. It minimizes the dispersion and allows a higher sampling frequency.
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Fig. 3. (a) Influence of the injection volume on the analytical response; 2.9510ÿ5 mol lÿ1 thiamine. (b) Calibration lines for 300, 600 and 1000 ml of injected volume.
3.3. Analytical features of the proposed method In the working conditions above established the calibration lines obtained according to the procedure described for the three injection volumes are shown in Fig. 3(b). The sensitivity is enhanced by increasing the injection volume. This increase can be calculated from the slope ratios (S) of the calibration graphs for the three volumes used in this study: S1000/3002.44; S600/300 1.74 and S1000/6001.44 (the subscripts represent the injection volume, ml). The sensitivity of the method proposed has been compared with that of the conventional FIA method (solution): for a 600 ml injection volume the sensitivity is more than 11 times higher than that of the corresponding solution method. The
sampling frequency decreased lightly as the injection volume increased. Table 1 summarizes the analytical ®gures of merit. The advantages obtained in sensitivity by increasing the injected sample volume are self-evident. 3.4. Effect of foreign species The effect of foreign species on the determination of 11 mg mlÿ1 of thiamine by the proposed procedure was carried out for 600 ml of sample volume. The species tested include other vitamins of the B complex and those commonly present along with thiamine. This study was undertaken by adding a known amount of foreign species to the thiamine hydrochloride solution of 11 mg mlÿ1. The highest concentration of
Table 1 Analytical features of the method Injected volume (ml)
Linear dynamic range (mg mlÿ1)
Equation (r)
RSD (%)
Detection limit (mg mlÿ1)
Sampling frequency (hÿ1)
300 600 1000
2.0±33.0 1.0±20.0 0.6±12.0
A0.0000.031c (r0.9999) A0.0170.054c (r0.9995) A0.0160.078c (r0.9994)
0.75a 0.89b 1.77c
0.42 0.25 0.16
18 16 14
Aabsorbance; cconcentration of thiamine; rregression coefficient. 28 mg mlÿ1 of thiamine. b 15 mg mlÿ1 of thiamine. c 9 mg mlÿ1 of thiamine. a
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Table 2 Effect of foreign species on the determination of 11 mg mlÿ1 of thiamine Foreign species
Tolerance mg mlÿ1 interferent/mg mlÿ1 thiamine
L-Carnitine, L-Lysine
>100 50 >40 20 18 16 10
Nicotinic acid, folic acid Biotine Ascorbic acid Vitamin B12 Vitamin B6 Nicotinamide, vitamin B2
foreign species tested was 1000 mg mlÿ1, except for biotine which was 440 mg mlÿ1. A foreign species is considered not to interfere if it produces an error not exceeding 5% in the determination of the analyte. Table 2 shows the results obtained. The method proposed shows a high tolerance level to all the species tested, which are frequently encoun-
tered along with thiamine including other vitamins and B-complex factors. The tolerance level to all these species is higher than the concentration usually found for them in pharmaceuticals containing thiamine. Moreover, the high tolerance level to the other B group vitamins (namely B12, B6 and B2) proves the high selectivity shown by the method for thiamine determination in the presence of these vitamins by using a spectrophotometric detector tunned in UV region. On the other hand, if the tolerance level of the method proposed to vitamins of the B group is compared with that found by conventional UV spectrophotometry at the same pH [17], we ®nd that the amount tolerated in the method developed in this paper is 72, 75 and 770 times higher than that shown by UV conventional spectrophotometry in homogeneous solution. Due to its cationic nature, vitamin B1 is selectively ®xed on the Sephadex CMC-25 cation exchanger (sensing microzone), and it strongly raises
Table 3 Determination of thiamine in pharmaceutical preparations Pharmaceutical preparation
b
Antineurine Astenolitc Hepacoban B12d Neurodavure Nervobionf Benervag Salvacolonh Trimetaboli a
Stated thiamine HCl content (mg)
125 50 (thiamine NOÿ 3) 50 250 100 (thiamine NOÿ 3) 300 48 600
Thiamine HCl founda (mgRSD (%)) Proposed method
Fluorimetric method
128.6 (0.9) 51.6 (1.7) 49 (2) 251.7 (0.6) 102 (1) 302.3 (0.8) 46.0 (0.8) 605.3 (0.7)
129.7 (1.8) 50.6 (0.9) 48 (1) 255.7 (0.2) 103 (2) 297.0 (0.7) 48 (2) 589.0 (0.5)
Data are based on the average obtained from three determinations. Injectable vials (from Tedec. Meiji. Farma., Madrid, Spain) containing thiamine 125 mg, pyridoxine 125 mg, cyanocobalamin 5000 mg, and didocaine 3 mg. c Drinking ampoules (from Elmu QuõÂmica FCA, Madrid, Spain) containing thiamine 50 mg, pyridoxine 50 mg, cyanocobalamin 50 mg, acetylglutamine 50 mg, inositol 30 mg, acetylaspartic acid 70 mg, carnitine 1000 mg, potassium aspartate 75 mg, magnesium aspartate 75 mg, ethanol 96% 400 mg, and excip. sodium saccharin 40 mg. d Injectable vials (from Bohm, Madrid, Spain) containing thiamine 50 mg, cyanocobalamin 30 mg, hepatic extract (B12 native) 60 mg, and calcium pantothenate 5 mg. e Capsules (from Belmac, Madrid, Spain) containing thiamine 250 mg, pyridoxine 250 mg, hydroxocobalamine acetate 25 mg, and excip. magnesium stearate csp 1 capsule. f Capsules (from E. Merck-Darmstadt Igoda, Barcelona, Spain) containing thiamine 100 mg, cyanocobalamin 1000 mg, and pyridoxine 100 mg. g Tablets (from Roche Nicholas, Barcelona, Spain) containing thiamine 300 mg. h Solid vial (from SALVAT, Barcelona, Spain) containing thiamine 48 mg, cyanocobalamin 144 mg, riboflavin 12 mg, pyridoxine 120 mg, calcium pantothenate 240 mg, nicotinamide 480 mg, lysine 7200 mg, excip. sodium saccharin 59.8 mg, and sucrose 6080 mg. i Syrup (from Uriach-Biohorm, Barcelona, Spain) containing in 100 ml thiamine 600 mg, pyridoxine 600 mg, cyanocobalamin 20 mg, methopine 35 mg, lysine hydrochloride 5 g, carnitine hydrochloride 75 mg, sorbitol 70% 20 g, and sucrose 45 g. b
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the tolerance level to the presence of the B vitamins (and of other B complex factors). 3.5. Analytical applications Pharmaceutical preparations containing thiamine hydrochloride, either alone or in mixtures with other vitamins of the B group or other ingredients of a different nature, were assayed by the method proposed for an injection volume of 600 ml; for the sake of comparison, the thiamine content of these pharmaceuticals was also determined by the AOAC ¯uorimetric method [18] (based on the oxidation of thiamine to thiochrome by potassium ferrocyanide) used as a reference method. In every pharmaceutical preparations the results were found to be very good (Table 3), thus con®rming the validity of the sensor proposed in this paper. 4. Conclusions The continuous ¯ow-through UV spectrophotometric sensor here developed for determination of thiamine, based on the transient retention of the analyte in the ¯ow cell and monitorization of its intrinsic UV absorbance, shows clear advantages with respect to the conventional UV spectrophotometric method, namely a very important improvement in sensitivity, selectivity, simplicity, and speed. These are achieved because the cation exchange nature of the solid support placed in the cell preconcentrates and selectively ®xes the cation thiamine in the detection region (active sensing microzone), thus preventing those non-cationic species present along with thiamine to interfere. Moreover, these features in combination with an FIA system in which the carrier itself regenerates the active microzone, make the sensor a cheap, continuous, simple, and reusable sensing device suitable for determination of thiamine in pharmaceuticals. If compared to the solid phase UV spec-
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trophotometric batch method previously developed [17], the present method is much faster and cheaper (because the sample, buffer solution and solid active support consumption are much smaller), much less tedious (for example, no weight of a known amount of resin is necessary for each determination), and more precise, although less sensitive due to the use of a much smaller volume of sample (up to 3000 times less). References [1] P. Ortega Barrales, M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz, Anal. Chim. Acta 360 (1998) 143. [2] M.L. FernaÂndez de CoÂrdova, A. Ruiz Medina, A. Molina DõÂaz, Fresenius' J. Anal. Chem. 357 (1997) 44. [3] L.F. CapitaÂn-Vallvey, M.D. FernaÂndez, I. De Orbe, R. Avidad, Analyst 120 (1995) 2421. [4] F. CapitaÂn, E.J. Alonso, R. Avidad, L.F. CapitaÂn-Vallvey, J.L. Vilchez, Anal. Chem. 65 (1993) 1336. [5] L.F. CapitaÂn-Vallvey, N. Navas Iglesias, I. De Orbe PayaÂ, R. Avidad, Talanta 43 (1996) 1457. [6] M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz, M.I. Pascual Reguera, L.F. CapitaÂn-Vallvey, Talanta 42 (1995) 1057. [7] K. Yoshimura, Anal. Chem. 59 (1987) 2922. [8] L.F. CapitaÂn-Vallvey, M.C. Valencia, G. MiroÂn, Anal. Chim. Acta 289 (1994) 365. [9] P. Richter, M.D. Luque de Castro, M. ValcaÂrcel, Anal. Lett. 25 (1992) 2279. [10] K. Yoshimura, S. Matsuoka, T. Tabuchi, H. Waki, Analyst 117 (1992) 189. [11] M. ValcaÂrcel, M.D. Luque de Castro, Flow-through (Bio)chemical Sensors, Chapter 2, Elsevier, Amsterdam, 1994. [12] R.M. Lin, D.J. Lin, A.L. Sun, Talanta 40 (1993) 381. [13] Z. Gong, Z. Zhang, Anal. Chim. Acta 339 (1997) 161. [14] Y. Wang, K. Wang, W. Liu, G. Shen, R. Yu, Analyst 122 (1997) 69. [15] D. Chen, M.D. Luque de Castro, M. ValcaÂrcel, Anal. Chim. Acta 261 (1992) 269. [16] M. ValcaÂrcel, M.D. Luque de Castro, Analyst 118 (1993) 593. [17] P. Ortega Barrales, M.L. FernaÂndez de CoÂrdova, A. Molina DõÂaz, Anal. Chem. 70 (1998) 271. [18] AOAC Official Methods of Analysis, vol. 2, 15th ed., Association of Official Analytical Chemists, Inc, Arlington, VA, 1990, p. 1050.