ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2009, том 64, № 12, с. 1265-1270
ОРИГИНАЛЬНЫЕ СТАТЬИ
YflK 543
DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE HPLC METHOD WITH POST-COLUMN IODINE-AZIDE REACTION FOR THE DETERMINATION OF THIOGUANINE
© 2009 r. Robert Zakrzewski
Department of Instrumental Analysis, University of Lodz Pomorska 163, 90-236 LodZ, Poland Received 09.01.2008; in final form 14.05.2009
A high-performance liquid chromatographic method of reversed-phase with a post-column iodine-azide reaction has been developed and validated for the determination of thioguanine. Isocratic elution was performed on a column of C18 using acetonitrile-water-sodium azide solution (1.5%; pH 6.5) 16 : 34 : 50 (v/v/v) as a mobile phase with How-rate of 0.5 mL/min. Monitoring of unreacted iodine in post-column iodine-azide reaction induced by thioguanine resulted in its detection at 350 nm. The method applied to thioguanine was linear within the scope of values 8-100 nM (r2 > 0.9988). The relative standard deviation (RSD < 4.2%) and the recovery (>96%) prove that the intra-day precision and the accuracy were satisfactory. The lower limits of detection (LLD) and quantification (LLQ) of thioguanine were established at the levels of 6 and 8 nM, respectively. The elaborated method was validated and applied to thiogua-nine determination in tablets.
2-Amino-1,7-dihydro-6H-purine-6-thione, a compound known as thioguanine, is closely related to guanine, a constituent of the nucleic acid. The structure and functions of thioguanine largely correspond to another drug mercaptopurine. Due to its chemotherapeutic properties, thioguanine is often applied in the treatment of some types of cancer [1], mostly leukaemia. The cytotoxic effect of the drug results mostly from the merge of the nucleotides with DNA and RNA in bone marrow cells in humans.
There are available reports on the use of several liquid chromatographic methods in the process of thioguanine determination in biological samples: blood plasma [2-4], urine [5], capillary blood [6], red blood cells [7], tissues [8], cerebrospinal fluid [9]. Yet, there are not reports on thioguanine quantification in tablets based on HPLC technique.
Therefore, the present study aims at the development and validation of a stability-indicating method that would facilitate thioguanine quantification using high-performance liquid chromatography. The immediate purpose for this work is to establish analytical routine and quality control procedures. In order to evoke a change in sensitivity, the iodine-azide reaction is chosen in this study as the method of detection. The scheme in Fig. 1 proves that the reaction is induced solely by sulfur(II) compounds. The procedure is based on the analyte separation on chromato-graphic column with the solution of sodium azide and ace-tonitrile in the mobile phase and subsequently on the measurement of iodine unreacted in the iodine-azide reaction. In the course of the process, a sulfur(II) compound undergoes selective induction. As a result of the absence of these compounds, iodine provides the source for the constant ab-sorbance, which is observed using HPLC system that provides azide ions from the mobile phase and with iodine so-
lution from post-column reagents. What is more, the signal decreases together with the presence of a sulfur(II) compound in the chromatographic band, as it is assigned to the consumption of iodine in the iodine-azide reaction. Hence, the negative peak is detected at X = 350 nm. The quantity of the peaks depends on the amount of the sulfur(II) compound. A flow diagram of the chromatographic system applied in the study is depicted in Fig. 2.
EXPERIMENTAL
Chemical and reagents. The grade of all chemicals was either analytical or HPLC. The suppliers of thiogua-nine, sodium azide, hydrochloric acid, sodium hydroxide, iodine, potassium iodide, and acetonitrile were: Aldrich (Steinheim, Germany), LAB-SCAN Analytical Sciences (Dublin, Ireland) or POCH (Gliwice, Poland). Thiogua-nine in tablets under the brand of Lanvis, manufactured by GlaxoSmithKline Export Ltd., UK, was labeled to contain 40 mg of thioguanine and was obtained commercially.
Preparation of working standard solution. The standards of the research included fresh, daily preparation of all the solutions. They were formulated with the use of deion-ized water with subsequent 15 min helium sparging. The preparation of the stock standard solution (1 mM) included
SH
2N- + I2
H2N^N^~nh
3N2 + 2I-
Fig. 1. Scheme of iodine-azide reaction induced with thioguanine.
Waste
Fig. 2. Diagram of flow system with iodine-azide detection. 1 - sodium azide solution, 2 - acetonitrile, 3 - water, 4 - pump, 5 - injector valve, 6 - analytical column, 7 - mixing tee, 8 - postcolumn reaction module, 9 - pump, 10 - iodine solution in potassium iodide solution, 11 - temperature control system, 12 - LC spectrophotometer, 13 - computer.
with dissolving 16.72 mg of thioguanine reference substance accurately weighed, in 1 mL of 1 M sodium hydroxide. The last step was the adjustment of the solution with water to 100 mL. The dilution of stock solution in the mobile phase resulted in the formulation of the thioguanine solution (0.1 ^M). Its stability was verified for the next 5 h in closed vial stored at room temperature.
Preparation of sample solutions. Twenty tablets were weighed in order to calculate the average weight. Afterwards, the tablets were crushed to a fine powder. The
amount corresponding to 4 mg was placed in the 100-mL volumetric flask and supplemented with 1 mL of 1 M sodium hydroxide and 10 mL of water. Prior to the dilution to volume with water, the flask was mechanically shaken for 15 min. Next, the solution was sipped through the quantitative filter paper, and 100 ^L of the obtained solution sample was diluted with mobile phase to 10 mL.
Preparation of solutions for chromatography. In a mobile phase, sodium azide in the amount of 7.5 g was dissolved in water with the supplementation of hydrochloric acid in order to obtain pH 6.5. Next, the solution was adjusted with water to the amount of 0.5 L. Afterwards, the solution (pH 6.5; 1.5% w/v) was mixed with acetonitrile and water in the proportion 50 : 16 : 34 (v/v/v) with the use of HPLC pump, as shown in Fig. 2.
A post-column reagent solution consisted of 6.3 g iodine and 20 g potassium iodide, dissolved and adjusted with water to 0.5 L. The obtained solution in the amount of 750 ^L was supplemented with 0.833 g of potassium iodide and diluted with water to 0.25 L.
The buffer solutions were adapted with the use of po-tentiometric titrations. The calibration of the titration system was accomplished with standard pH solutions. All reagents were tested for stability for the purpose of unattended analysis.
Apparatus and chromatographic conditions. The
equipment used for the chromatographic separation was Waters liquid chromatographic system equipped with Multisolvent Delivery System Model 600E, 717plus au-tosampler, and a variable wavelength LC spectrophotome-ter (2487 Dual A). The chromatographic separation was performed at ambient temperature on an analytical column, Symmetry C18 (150 mm x 3.9 mm i.d., 5 ^m, Waters). A flow rate of 0.5 mL/min at ambient temperature was chosen for the mobile phase mixture of acetonitrile-1.5% sodium azide, pH 6.5-water (16 : 50 : 34, v/v/v).
The iodine-azide post-column reaction was accomplished on Waters system with the support of Reagent Manager as a single-piston, pulsedampened pumping system. The post-column reagent (the mixture of 0.3 mM iodine solution in 20 mM potassium iodide) was delivered to the Post-column Reaction Module (the reaction tube, 6 m x x 0.46 mm i.d.) at the flow-rate of 0.2 mL/min with the use of Temperature Control System. The volume of injection was 20 ^L and the detection was executed at a wavelength of A = 350 nm. The response was monitored with Empower™ software (Waters). A schematic diagram and optimum conditions for the thioguanine separation by HPLC are presented in Fig. 2 and Table 1, respectively. The results of thioguanine quantitation by the post-column reaction are reported in Table 2.
Linearity. Thioguanine stock solution of 1 ^M was diluted with mobile phase in appropriate amounts in order to provide concentrations of 8, 10, 30, 40, 50, 60, 800, 100 nM. Each concentration was developed in fortifying. The evaluation of the linearity was performed with the use of linear regression analysis, which by the method of least square regression.
Precision. The process of precision determination was accomplished through the analysis of three sample thioguanine solutions on the same day for intra-day precision (repeatability) and on three days for inter-day precision (intermediate precision). As a result, the relative standard deviation (RSD%) was established.
Accuracy. Accuracy was performed in the form of recovery test by spiking the sample solution with the known amounts of thioguanine. The test was conducted in the analysis of three different solutions, each in four replicates, containing 8.0, 40.0 and 100 nM. The solutions represented the low, medium and high concentration of the linearity concentration range, respectively.
RESULTS AND DISCUSSION
Method development. A reversed-phased HPLC method with post-column iodine-azide reaction was chosen for the purpose of quantification of thioguanine in tablets. The properties of thioguanine governed the mobile phase and the flow-rate selection. The compound induced the aforementioned reaction and influenced on other variables of the study: quality of the chromatographic separation (peak symmetry, number of theoretical plates, peak tailing, and resolution), run time, ease of preparation, and cost. The separations were acceptable, with a retention time of 4.1 min for thioguanine, with the use of the mobile phase of acetonitrile: sodium azide solution (1.5%; pH 6.5): water 16 : 50 : 34 (v/v/v), at 0.5 mL/min. These conditions o
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.