Voltammetric determination of leucovorin in pharmaceutical samples
PAVLÍNA MARTINKOVÁ a, *, BARBORA KRÄNKOVÁ a, RENATA ŠELEŠOVSKÁ a, LENKA JANÍKOVÁ a
a University of Pardubice, Faculty of Chemical Technology, Institute of Environmental and Chemical Engineering, Studentská 573, 532 10 Pardubice, Czech Republic, st36636@student.upce.cz

Abstract
Voltammetric behavior of leucovorin has been investigated for the first time using boron-doped diamond electrode (BDDE) and the method for its determination was developed. Leucovorin provides two well-developed oxidation peaks at about +900 and +1450 mV (pH 5) on BDDE. Britton-Robinson buffer (pH 3.0) was chosen as an optimal supporting electrolyte for its determination using differential pulse voltammetry (DPV) with optimized parameters. Limit of detection 1.5×10−8 mol L−1 was reached for leucovorin determination and linear dynamic range 1.5×10−7-2.5×10−5 M was obtained. Relative standard deviation of repeated measurements (RSDM(11) = 0.7 %) and relative standard deviations of repeated determinations (RSDD(5) < 3.0 %) were calculated to confirm very good results. Applicability of the proposed method was verified by an analysis of a pharmaceutical preparation. 

Key words: Leucovorin, Oxidation, Boron-doped diamond electrode, Differential pulse voltammetry.

1. Introduction
Leucovorin (LV, folinic acid, Fig. 1A) is one of reduced derivatives of folic acid (FA, Fig. 1B). Folates participate in the transfer of activated single carbon units which are involved in purine synthesis. Purines are necessary for the formation of nucleic acids and thus to restore and cell division [1]. On the contrary, antifolate methotrexate (MTX) blocks the purine synthesis in all cells and prohibits their further division. Due to the rapid division of cancer cells, their damage is the greatest. MTX is, therefore, an important chemotherapeutic drug [1, 2]. LV acts as an antidote of MTX [3, 4]. It can be also administered in high doses as co-medication to enhance the effectiveness of the chemotherapeutic agent 5-fluorouracil (5-FU) in the treatment of colorectal and gastric carcinoma [5]. 
	Although LV is electrochemically active compound, only a few papers dealing with its electrochemical behavior or with its voltammetric determination have been published, and, moreover, only with the use of mercury based electrodes. Allen et al. described the polarographic behavior of LV using dropping mercury electrode (DME) [6]. Stejskal et al. used hanging mercury drop electrode (HMDE) and proposed the method for LV determination [7]. Finally, Šelešovská et al. described voltammetric behavior of LV on mercury meniscus modified and polished silver solid amalgam electrodes (m-, p-AgSAE) and proposed the method for its determination with the limits of detection (LOD) 2.2×10−8 and 5.0×10−8 mol L−1 [8].
	This work represents a contribution to this area focused on voltammetric behavior of LV on BDDE. This electrode was introduced for electroanalytical chemistry in the early nineties of the twentieth century [9, 10]. In terms of electroanalytical chemistry, the wide potential window about 3 V is the greatest advantage of BDDEs. Furthermore, these electrodes exhibit the low noise, low adsorption on the surface, and minimal problems with passivation [11]. BDDEs are most often used in the analysis of organic compounds and our research group has also published a number of papers dealing with the development of voltammetric methods for determination of various pesticides and drugs [12-17].
	
2. Experimental
2.1 Reagents and chemicals
The standard solution of 1×10−3 mol L−1 LV (Sigma-Aldrich, Czech Republic) was prepared by dissolution of the appropriate amount in methanol (Penta, Czech Republic) and then was stored in the dark in a refrigerator. Britton-Robinson buffer (BRB) of a pH value from 2 to 12 was prepared from an alkaline component of 0.2 mol L−1 NaOH (Lachema, Brno, Czech Republic) and an acidic component consisting of H3PO4, H3BO3 and H3COOH (all Lachema, Brno, Czech Republic) of the same concentration (0.04 mol L−1). The electrolytes based on various concentrations of H2SO4 were prepared from 96 % H2SO4 (Penta, Czech Republic). Pharmaceutical preparation “Leucovorin CA LACHEMA 10” originated from Pliva-Lachema, Brno, Czech Republic.
2.2 Instrumentation
All voltammetric measurements were performed by computer controlled Eco-Tribo Polarograph [18] (Polaro-Sensors, Prague, Czech Republic) with POLAR.PRO software (version 5.1). All measurements were provided in a three electrodes set up, where commercially purchased BDDE (7.07 mm2) with B/C ratio during deposition step of 1000 ppm (declared by producer Windsor Scientific, United Kingdom) served as the working electrode. Saturated silver/silver chloride electrode (Ag/AgCl) was used as a reference and platinum wire as an auxiliary electrode (both Monokrystaly, Turnov, Czech Republic). The measurements were performed at laboratory temperature (23±2°C). Oxygen was not removed from the measured solutions. 
Procedures
BDDE was activated at the beginning of each working day in solution of 0.5 mol L−1 H2SO4 by insertion of cathodic potential value of −2000 mV for 60 s and anodic potential value of +2000 mV for 60 s as well. Finally, 20 cycles from −1000 to +2000 mV (100 mV s−1) were applied. Regeneration of the electrode surface between measurements was realized by insertion of potential +2000 mV for 5 s. Cyclic voltammetry (CV) was used at first for the examination of voltammetric behavior of LV on BDDE in dependence of pH of the supporting electrolyte and scan rate (v). Measurements were carried out in the potential range from −1000 mV to +2000 mV with scan rate of 100 mV s−1. The influence of scan rate was recorded in the range from 25 to 500 mV s−1. DPV with pulse height of +50 mV, pulse width of 20 ms, and scan rate 40 mV s−1 was utilized for determination of LV. DPV peaks were evaluated from the straight line connecting the minima before and after the peak (tangent to the curve joining the beginning and end of a given peak). 

3. Results and discussion
Voltammetric behavior of LV was studied using CV. It was found that LV provides two oxidation peaks in wide range of pH as it can be seen in Fig. 2. From the inserted dependence of peak height (Ip) on pH it is obvious that the highest current responses were obtained in BRB of pH 2. On the other hand, the first LV signal, which appeared to be more appropriate for analytical use, was badly repeatable in this acidic medium (RSD11 = 8.5 %). Therefore, BRB of pH 3 (RSD11 = 1.9 %) was chosen as a supporting electrolyte for the following LV analyses. The linear dependence of Ip on square root of scan rate was obtained using CV for both LV responses which corresponds with diffusion-controlled processes.
	DPV was utilized for the development of method for LV determination. Its parameters like scan rate (v), pulse height, and pulse width were optimized (summarized in experimental part) as well as the conditions of BDDE surface regeneration. This technique was then applied for LV determination in model solutions using BDDE. An example of the obtained concentration dependence for LV in range from 2.99×10−7 to 2.45×10−5 mol L−1 is presented in Fig. 3. It demonstrates very wide linear dynamic range of this method which was from 1.5×10−7 to 2.5×10−5 mol L−1. The following statistical parameters of the proposed method were obtained, namely LOD 1.5×10−8 mol L−1 and limit of quantification (LOQ) 5.0×10−8 mol L−1. The repeatability of LV determination was tested by its 5 times repeated determinations for various concentration levels of analyte (1.0×10−5, 3.0×10−6, and 3.0×10−7 mol L−1). The obtained results summarized in Table 1 are accurate and prove very good repeatability of proposed method of LV determination (RSD5 < 2.6 %).
	The applicability of the developed voltammetric method was veriﬁed by the analysis of the pharmaceutical sample, namely the powder for preparation of injection solution, with declared content 10 mg LV per vial. Injection solution was prepared by dissolving of LV powder in distilled water according to the producer instruction. The content of LV was determined ﬁve times in this injection solution using the standard addition method. The determined amount of 10.08±0.12 mg LV per vial (recovery 98.7-102.8 %, RSD5 = 1.81 %) coincided with the declared one. Therefore, it could be concluded that the designed method is suitable also for analysis of pharmaceutical samples without any complicated procedure only after simple dilution and it can be applied for quality control of pharmaceuticals.

4. Conclusions
Voltammetric behavior of LV on BDDE using CV was described in the present paper. It was found that two anodic signals belonging to the oxidation of LV could be recorded in wide pH range. The highest stable signal was recorded in BRB of pH 3, which was applied in all further analysis. The first oxidation signal was found suitable for analytical purposes and, thus, after its optimization DPV was successfully applied to the analysis of model solutions as well as pharmaceutical sample. 

Acknowledgements
This work was supported by the grant project of The Czech Science Foundation (project No. 17-03868S) and by The University of Pardubice (projects No. SGSFChT_2017_002 and SD373001/82/30350(2016)).

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Figures

B

A

Fig. 1. Structural formula of leucovorin (a) and folic acid (B)

Fig. 2. Cyclic voltammogram of leucovorin obtained on BDDE. Method: CV, BRB (pH 5.0), Ein = Efin= 0 mV, Eswitch = +2000 mV, v = 100 mV s−1, cLV = 5×10−5 mol L−1; Inset: Dependences of Ip on pH (0.05 mol L−1 H2SO4 (pH 1), BRB (pH 2-12)).

Fig. 3. DP voltammograms of LV obtained on BDDE in dependence on concentration. Method: DPV, BRB (pH 3), Ein = 0 mV, Efin= 1200 mV, v = 40 mV s−1, pulse height = 50 mV, pulse width = 20 ms, cLV = 2.99×10−7-2.45×10−5 mol L−1; Inset: Dependence of Ip on cLV for the peak 1.

Tables
Table 1. Results of repeated determination of LV in model solutions
Added [mol L−1]	Found [mol L−1]	Recovery [%]	RSD5 [%]	
1.0×10−5	(1.010±0.017)×10−6	98.0-105.0	2.57	
3.0×10−6	(3.030±0.035)×10−6	98.3-102.6	1.74	
3.0×10−7	(3.000±0.029)×10−7	99.0-102.6	1.46