A novel online coupling of ion selective electrode with the flow injection system for the determination of vitamin B1

A simple, fast, selective of a new flow injection analysis method coupled with potentiometric detection was used to determine vitamin B1 in pharmaceutical formulations via the prepared new selective membranes. Two electrodes were constructed for the determination of vitamin B1 based on the ion-pair vitamin B1phosphotungestic acid (B1-PTA) in a poly (vinyl chloride) supported with a plasticized di-butyl phthalate (DBPH) and di-butyl phosphate (DBP). Applications of these ion selective electrodes for the determination of vitamin B1 in the pharmaceutical preparations for batch and flow injection systems were described. The ion selective membrane exhibited a near-Nernstian slope values 56.88 and 58.53 mV / decade, with the linear dynamic range of vitamin B1 was 5 x 10 -5 1 x 10 -2 and 1 x 10 4 -1 x 10 -2 mol.L -1 , in batch and FIA, respectively. The limit of detection was 3.5 x 10 5 and 9.5 x 10 -5 mol.L -1 , with the percentage linearity 98.85 and 95.22 in batch and FIA, respectively. The suggested ion selective electrode has been utilized perfection in the determination of vitamin B1 in pharmaceutical formulations using batch and flow injection system, respectively.


Introduction
Vitamin B 1 , also known as thiamine hydrochloride is {3-[(4-Amino-2-methylpyrimidin-5-yl) methyl]-5-(2-hydroxyethyl)-4methylthiazolium chloride hydrochloride} ( Figure 1) [1]. A deficiency of this vitamin B 1 causes many diseases in adults and children with high intake of white rice or food containing anti-thiamine factorsm, e.g. beriberi [2], and epilepsy [3]. Many of analytical methods are available for the determination of vitamin B 1 either alone or in conjunction with various drugs in pharmaceutical formulations and biological fluids using calorimetric [4], spectrophotometry [5][6][7], electrochemistry [8], fluorimetric [9], chromatography [10][11][12][13][14][15], chemiluminescence [16]. Potentiometric methods are more productive in innovations in view of the more possibilities for the design of the electrodes that can easily be converted into flow-through electrodes via passed in the flow cell. Consequently, the focus of this work includes two points. Firstly, is a building up a new online multi semi-automated system that is capable to determine pharmaceuticals preparations which depends on the use of selective membranes and the possibility of this system for measuring one of these pharmaceuticals, or group of pharmaceuticals in the mixture by selectively choosing the kind of the mode. Secondly, new ion selective membranes for vitamin B 1 based on ionpair of vitamin B 1 with PTA were prepared using DBPH and DBP as plasticizers.

Material and methods: Reagents and materials
The thiamine hydrochloride standard (C 12 H 17 ClN 4 OS.HCl) was obtained from Samara drug industry. The pharmaceutical formulations samavit (tablet) and thiamine injection (ampoule) were supplied by Samira-Iraq and Beijig-china companies, respectively. Phosphotungestic acid was obtained from Merck. Di-butylphthalate (C 16

Apparatus Complementary parts in batch-system
Microprocessor based Bench pH/MV/C Meter (model pH211, HANNA instruments (Romania)) was used to measure the potentiometric signals. Calomel reference (USA) and silver-silver chloride (Ag/AgCl) electrodes were used as external reference and an internal reference, respectively. Portable pH/ORP Meter (model HI 9125, HANNA instruments (USA)) was used for adjusting pH of solutions. UV/VIS spectrophotometer model SP-3000 plus, Optima INC, (Japan) was used for dispersion calculations.

Complementary parts in flow-system
A new flow injection system was consisted of peristaltic pump (model Gilson 72Rue Gambetta B.P. 45 (France)), and used with one line. An injection valve was used to inject sample volume in the carrier stream (distilled water line). Silicon rubber and Teflon tubing used for connecting tubes (I.D: 0.5, 0.7, 1 mm). Four Teflon electrodes (homemade) have been utilized. Flow cell (homemade) made from poly methyl methacrylate (PMMA) used for entrapment of the Teflon electrode, and also facilitate the flow of solutions. A new electronic system (homemade) that based on field effect transistor (FET) was used to record the results.

Preparation of ion-pair
The ion-pair of B 1 -PTA was synthesized by mixing 0.01 mol.L -1 of vitamin B 1 with 0.01 mol.L -1 of PTA. The obtained precipitated was filtered and washed with distilled water, then dried in room temperate. This ion-pair was used for synthesis of electrode membrane.

Synthesis of membrane
Two membranes were prepared by mixing an amount of ion-pair (B 1 -PTA) with DBP or DBPH and PVC which dissolved in 8-10 mL of THF. The membrane was cut equal to the external diameter at the edge end of the electrode. These membranes were used as sensors for the determination of vitamin B 1 . It is worth to be mentioned the membrane was immersed in standard of vitamin B 1 in 2 hours before using for measurements.

Construction of calibration curve
In batch mode, the concentrations range of vitamin B 1 were prepared from 1×10 -6 to 1×10 -2 mol.L -1 and recorded the measurements potential for each concentration, then plotted between potential and concentration. In FIA mode, range of concentrations 1×10 -6 to 1×10 -2 mol.L -1 of vitamin B 1 was injected to the flow system under optimum conditions from flow rate and volume injection (using the third site in new flow injection for this research). The obtained peak heights were recorded and used to plot the calibration curve.

Potentiometric
analysis for determination of vitamin B 1 Direct method was consisted of the measured response for sample solution and read the concentration from the calibration graph that prepared from vitamin B 1 standard solutions. In the standard addition method, 0.1 mL of vitamin B 1 standard solution (0.01) was added to 50 mL of the same sample pure or pharmaceutical formulations.The change in electrode response was recorded and used to calculate the concentration of sample by following Equation: Where C U, C S the concentration of unknown and standard solution, respectively, V U, V S are the volume of unknown and standard solution, respectively, S: the slope of electrode, ΔE: the potential difference [17].  7.0 × 10 -5 3.5 × 10 -5 **If the: assumption was mode. Null Hypothesis H o : ρ=0 (no correlationnon linear). Alternative Hypothesis A: ρ≠ 0 (significant correlationsignificant linear).It can be seen that the calculated practically found value of t is larger than the critical tabulated value at 95% confidence interval │t│> t α/2 (n-2) therefore H o is rejected against A. i:e linearity is accepted for the range given.* n=3.

Results and Discussion:
Optimization of ISE response in batch condition Influence of plasticizers on the electrode response Two plasticizers DBPH and DBP were investigated (Table 1). Table 1 exhibits that DBPH is the best of the plasticizers examined due to high mixing between plasticizer and PVC that leads to production of homogeneous membrane. Therefore DBPH plasticizer gave near-Nernstian response. While DBP shows the poor sensitivities on the electrode response. The reason for this must be to high viscosity of the plasticizer which decreases the ionexchange process.

Influence of pH
The effect of pH of the vitamin B 1 solution (1×10 -3 , 1×10 -4 ) mol.L -1 on B 1 -PTA electrode and DBPH plasticizer was studied by adding dilute solution of hydrochloric acid (0.1 mol.L -1 ) and sodium hydroxide (0.1 mol.L -1 ). Figure  3 illustrates the B 1 -PTA electrode can be used in the pH range of 4-7 with good sensitivity. It should be noted increasing pH (pH >8) leads to decrease electrode response due to that vitamin B 1 was insoluble in basic solution. The electrode response increases in strong acid (pH < 3) solution. This indicates that ion-pair (B 1 -PTA) might probably respond to hydrogen ions.

Response time
The response time was measured for the vitamin B 1 electrode based on DBPH for two concentrations (1×10 -2 , 1×10 -5 ) mol.L -1 as shown in the Figure  4. The values of response time increase as the concentration decrease. This is attributed to the need for more time to reach the equilibrium between the ion-

Lifetime of the electrode
The lifetime of the B 1 -PTA electrode was investigated by accomplishing calibration curve ( Figure  5) as a function of immersing time. The results show that the slope of the calibration curve was 56.89 mV decade -1 after one week immersing in 1×10 -2 mol.L -1 , then the slope decreased, reaching 56.49, 53.62, 50.59 and 45.46 mV decade -1 after 3, 5, 6, 7 weeks, respectively. Theoretically, the lifetime of this electrode is terminated in the fifth week of soaking, but practically, the electrode is fit for the use on the lower slope value. After this time, the electrode becomes less sensitive toward vitamin B 1 , which may be due to gradual leach of the ion-pair from the membranes to the external solution.

FIA conditions
A new manifold system consists of four sites of the flow system. Each site contains one electrode, and each electrode with drug membrane that is different from the other electrode. The flow measurements were accomplished in a one line system, the analyte is injected via the injection valves passes through the pump to the sites of each of the four electrodes. In this research, vitamin B 1 electrode that in the third site was studied. The dispersion effect and site effect on vitamin B 1 electrode response were studied. The dispersion of the third site is equal 2.13.

Influence of flow rate
This factor was studied by varying flow rates (0.54-2.04) mL.min -1 at constant injection volume equal 1 mL and vitamin B 1 concentration (1×10 -3 ) mol L -1 . The results of variations of the flow rate show that increasing the flow rate leads to an increase in response. The effect of flow rate on width base (∆t B ) was also studied. In the low flow rate, the travelled distance of the sample increases because of the dispersion in sample segment and the long distance between the site of the electrode and the injection point. While in the high flow rate, the travelled distance of the sample is decreased, due to fast entry and departure from the site as shown in

Influence of injection volume
The variations of the sample volumes (0.5-2.5 mL) were injected of vitamin B 1 (1×10 -3 mol.L -1 ) at a constant flow rate of 2.04 mL.min -1 . It should be noted that the large volume of vitamin B 1 is injected leads to an increase in response and dispersion (irregular response, broad signal) as shown in Figure 7. In addition the increased sample volume will consume large amount of the vitamin B 1 . At the low injected sample volume of vitamin B 1 leads to low response. The influence of the sample volume effect on ∆t B was studied. Increasing the injected volume leads to an increase of the travelled distance that causes the dispersion in sample segment (irregular response, broad signal). At low injecting volumes of drug leads to low response. Therefore the injected volume of 1 mL was chosen as the best sample volume with better response.

Influence of buffer pH
The effect of buffer on the vitamin B 1 -PTA electrode was studied in the range of pH 1-9 using hydrochloride acid/potassium chloride buffer (pH=1-2), sodium acetate/acetic acid buffer (pH=3-6) and disodium hydrogen phosphate/ hydrochloride acid (pH=7-9) [18][19] at (1×10 -3 and 1×10 -4 ) mol.L -1 of vitamin B 1 , 2.04 mL min -1 of flow rate and 1 mL sample volume as shown in the Figure 8. It observed that there are no significant changes in potential in the pH range of 4 to7, indicating that the electrode works in this range. At low pH values the response of the electrode can be noticed that it increases with lower values of pH. At the higher pH value the electrode might be poisoned, giving the irregular responses due to the precipitation of the vitamin B 1 .

Calibration curve
The performance characteristics are, slope 58.53mV/decade with a linear range (1×10 -4 -1×10 -2 ) mol.L -1 and detection limit 9.5×10 -5 mol.L -1 at the optimum conditions (flow rate 2.04 mL.min -1 ,volume injection 1 mL and a carrier stream of buffer solution (pH 6)) were studied in the third site of the manifold system (Table 2). Figure (9) shows the calibration curve for vitamin B 1 electrode.

Interfering ions effect
The influence of some inorganic cations on vitamin B 1 electrode was studied. The selectivity coefficients were measured using a separate solution method as Equation 2 (Nicolsky-Eisenman Equation) [20].  Table 3.It was noticed from  Table 3, the selectivity coefficient values for monovalent cations is higher than the values for di and trivalent cations interferences, which might be due to differences in ionic size, mobility and permeability, so that the monovalent ion interfere with the electrode response more than the di and trivalent cations. **t-test calculated 7.7305 Detection limit (mol.L -1 ) 9.5 × 10 -5 **If the: assumption was mode. Null Hypothesis H o : ρ=0 (no correlationnon linear). Alternative Hypothesis A: ρ≠ 0 (significant correlationsignificant linear).It can be seen that the calculated practically found value of t is larger than the critical tabulated value at 95% confidence interval │t│> t α/2 (n-2) therefore H o is rejected against A. i:e linearity is accepted for the range given. *n=3

Analytical application
Potentiometric techniques were applied in investigating electrode to determination of vitamin B 1 in pharmaceutical preparations samavit tablet and thiamine injection using direct and standard addition methods in batch and FIA conditions. The results were summed up in the Table 4.  Table 4 exhibits it can be seen that the obtained results from standard addition method have good precision and accuracy compared with the direct potentiometry method. This might be due to the effect of cancelling the dilution factor while preparation of solution in the normal working procedure compared to the standard addition method and, therefore, the results in less error.

System effect of different location for vitamins B 1 electrodes at different sites in the newly developed manifold
Extended experiments were conducted to establish the kind of behavior that the electrode response can act based on the design of the system in establishing all factors that might affect the whole measurements of the electrode response. The main factors that will be studied from preliminary observation of how the system works it was concluded that peak height,∆t B , t 0 -t 1 , are the most important factors. The experiments were carried out in site one which is defined for vitamin B 1 electrode; the three factors were studied at this site; followed by transferring the electrode to the second site; where the sample passes directly to this site without passing to the first site. At this point all the three factors are studied, followed by transferring the vitamin B 1 electrode to the third site, where the sample segment passes through to this site without passing through to the first site or even to the second site. After all, the three factors were measured ; the electrode was transferred to the fourth site and the sample segment when injected will pass directly tothe fourth site without passing the trend of the three previous sites, all these are shown in Figures  (10a, b) and (11) which indicate clearly the flow of fluid (i.e sample + caier stream). This kind of methodology adopted here will take into account that in case of maintenance of whatever the nature is; single electrode techniques will still can proceed without difficulty of complete stoppage. It was noticed that the obtained value represents a decrease in both 1,2 sites while there is an increase at site 4. It can be seen from Figure (10a) related to peak height variables that there is in general a slight decrease in all responses for each replaceable electrode site compared to its original location. Comparing ∆t B values. It was noticed from Figure (10b) related to system effect study that there is as a trend of a systematic increase in ∆t B . It can be attributed to delay of the arrival sample segments to the electrode site. Relatively according to its allocation. While for arrival time i.e t 0 -t 1 there is a logic increase or decrease in t 0 -t 1 according to its position in the system as shown Figure 11.

Conclusion :
The present work demonstrates the construction of new vitamin B 1 and PVC. The construction electrodes work in a satisfactory manner in the concentration range from 5 x 10 -5 to 1 x 10 -2 with a good operational lifetime and the response time. The best vitamin B 1 electrode was based on DBPH. A new FIA system is simple to construct and operate was built up . The construction electrodes were successfully applied for determination vitamin B 1 in pure solution and in pharmaceutical preparations using batch and FIA. The obtained results show the propose method is simple and rapid, with good accuracy.