DEVELOPMENT AND VALIDATION OF HPLC / UV-PROCEDURES OF SECNIDAZOLE DETERMINATION IN BLOOD AND URINE ©

Мета. Система ВЕРХ-аналізатора MiLiChrome® A-02 широко використовується в українських лабораторіях судово-медичної токсикології. Мета – застосувати систему ВЕРХ-аналізатора для кількісного визначення секнідазолу в біологічних рідинах і провести валідацію розроблених методик. Методи. Пробопідготовка крові і сечі здійснювалася трьома способами: 1) рідко-рідинна екстракція органічними розчинниками, що не змішуються з водою, 2) екстракція амфіфільними розчинниками і висолювання амоній сульфатом, 3) комплексне застосування рідко-рідинної екстракції органічними розчинниками, що не змішуються з водою, і амфіфільними розчинниками з наступним висолюванням. Умови хроматографування: колонка – 275 мм, ProntoSIL 120-5-C18 AQ, 5 мкм; температура – 40°С; швидкість потоку – 100 мкл/хв.; елюент А – 0,2 М LiClO4 – 0,005 М HClO4; елюент Б – ацетонітрил; режим елюювання – лінійний градієнт; детектування – УФ, 277 нм; об’єм проби – 2 мкл. Результати. Валідація всіх розроблених методик проводилась за такими параметрами, як специфічність, ступінь ізолювання, лінійність, правильність і прецизійність у варіанті методу стандарту. Результати аналізу показали відсутність піків з часом утримування, який співпадає з часом утримування секнідазолу, на хроматограмах blank-зразків для всіх варіантів способів пробопідготовки. Всі процедури пробопідготовки показали високу ефективність ізолювання секнідазолу як для крові, так і для сечі (на рівні 90 %). Всі розглянуті процедури характеризуються прийнятними параметрами лінійності, within-run і between-run правильності і прецизійності. Висновки. Розроблено комплекс ВЕРХ-методик кількісного визначення секнідазолу в крові і сечі. Проведено валідацію розроблених методик; застосування ізопропанолу в кислому середовищі оптимальне для пробопідготовки біологічних рідин Ключові слова: секнідазол, високоефективна рідинна хроматографія, кров, сеча, пробопідготовка, валідація, метод стандарту

out by extraction with ethyl acetate [13]. Two HPLC/UV-procedures more was described for secnidazole quantification in human plasma with application of such columns as Dikma ODS-C18 [14] and Bondex C18 [15]; mobile phase was the mixture of water and acetonitrile in different ratio (UV-detection -320 nm); the range of application was 0.25 -50 g/ml and 0.5-50 g/ml respectively. The method of HPLC/MS/MS was described for secnidazole determination in bees wax [16] and muscle samples [17]; such columns as Luna PFP 2150 mm, 3 m [16] and Kinetex XB-C18 2.1100 mm, 1.7 m [17] was used; mobile phase was the mixture of 0.01 % acetic acid in acetonitrile and 0.01 % acetic acid in water [16] and the mixture of 0.01 % formic acid in acetonitrile and 0.01 % formic acid in water [17]; the range of application was 0.2 -20 g/ml and 0.5 -10 g/ml respectively.

The field of research considering the general problem, which is described in the article
The system of HPLC-analyzer MiLiChrome® A-02 [18] is widely used in Ukrainian laboratories of forensic toxicology. Previously [19] we developed the HPLC/UV-procedure for secnidazole quantitative determination using this chromatographic system, carried out its validation by model solutions and showed the possibility for application in analytical toxicology.

Formulation of goals (tasks) of article
The purpose of present work is to apply the described HPLC/UV-procedure for secnidazole quantitative determination in biological liquids (whole blood and urine) using different types of sample preparation procedures, carry out its validation by matrix samples in the variant of the method of standard and prove the acceptability of application for forensic toxicology.

Presentation of the main research material (methods and objects) with the justification of the results
Secnidazole was of pharmacopoeial purity (99.7 %) and obtained from the pharmaceutical company TOV «Zdorovye».
The procedure of calibration and model samples preparation of blood and urine is presented on Fig. 1  The stock solutions 3 (100 μg/ml) were prepared by dissolving 50.0 mg of secnidazole in 0.01 mole/l hydrochloric acid solution and the solutions were diluted to 500.0 ml with the same solvent. The reference solution (8 μg/mL) was prepared by diluting 4.00 ml of the stock solution 3 to 50.0 ml with 0.01 mole/l hydrochloric acid solution.
The stock solution 3 was diluted with 0.01 mole/l hydrochloric acid solution to prepare the model solutions 1-4 having concentrations of 2; 4; 8 and 14 μg/ml respectively.
The procedures of sample preparation of biological liquids for secnidazole determination are presented on  Procedure 1: 25.00 ml of blood or urine was acidified with 6 M hydrochloric acid solution to pH2. The mixture was vortexed for 1 hour, then centrifuged for 5 minutes at 5000 rpm. The supernatant (рН should be less than 2) was extracted with 10.00 ml of chloroform triply (chloroform extracts were separated and removed).
The aqueous layer was salted-out by adding ammonium sulphate till stopping its dissolution, then neutralised with 25 % ammonia solution and extracted with 10.00 ml of the mixture of chloroform and 2-propanol (80:20) triply. The obtained organic extracts were separated, filtered through the paper filter with 1 g of sodium sulphate anhydrous into the measuring flask with the capacity of 50.0 ml, and diluted to the volume with chloroform.
Procedure 2: 25.00 ml of blood or urine was acidified with 6 M hydrochloric acid solution to pH2. The mixture was vortexed for 1 hour, then centrifuged for 5 minutes at 5000 rpm. The supernatant (рН should be less than 2) was extracted with 10.00 ml of chloroform triply (chloroform extracts were separated and removed).
The aqueous layer was processed twice with 10.00 ml of acetonitrile and vortexed for 1 hour each time. After adding 2 g of ammonium sulphate the mixture was filtered through the paper filter (wetted with acetonitrile) into the separating funnel and salted-out by adding ammonium sulphate till stopping its dissolution. Top organic layer was separated, filtered through the paper filter with 1 g of sodium sulphate anhydrous into the measuring flask with the capacity of 50.0 ml, and diluted to the volume with acetonitrile.
Procedure 2: 25.00 ml of blood or urine was acidified with 6 M hydrochloric acid solution to pH2. The mixture was vortexed for 1 hour, then centrifuged for 5 minutes at 5000 rpm. The supernatant (рН should be less than 2) was processed twice with 10.00 ml of acetonitrile and vortexed for 1 hour each time. After adding 2 g of ammonium sulphate the mixture was filtered through the paper filter (wetted with acetonitrile) into the separating funnel and salted-out by adding ammonium sulphate till stopping its dissolution. Top organic layer was separated, filtered through the paper filter with 1 g of sodium sulphate anhydrous into the measuring flask with the capacity of 50.0 ml, and diluted to the volume with acetonitrile.
The model and calibration samples, and also blank-samples were analysed for each procedure of sample preparation; the blank-samples were prepared in the following way: 5 samples (10.00 ml) of the matrix obtained from the different sources, in 1.00 ml of distilled water was added into them.
The HPLC/UV-method for secnidazole determination was proposed by authors before [19] and its specificity in relation to other 5-nitroimidazoles was shown.
We have carried out validation of all developed procedures by such parameters as specificity, recovery, linearity, accuracy, repeatability and intermediate precision according to the approaches offered by us in the variant of the method of standard [20][21][22][23].
The validation procedure foresees application of the normalized coordinates. For normalization of the obtained experimental data the reference solution with the concentration of analyte corresponded to its concentration in the end solution to be analysed under the condition of zero losses for the point of 100 % in the normalized coordinates is used. The peak area for reference solution is corrected taking into account the value of recovery R, which significance and value has been showed at the preliminary stage of validation, and is used for normalization of peak areas for the model samples.
The methods validation has been carried out using model samples -the determination procedure and acceptability criteria [20][21][22][23] are presented at Fig. 3.
The results of analysis have shown the absence of peaks with the retention time, which is coincident with (or near to) the secnidazole retention time, on the chromatograms of blank-samples for all variants of procedures of analyte isolation from biological liquids that points to the conclusion about acceptable specificity of the developed methods as for the components of biological matrix.
The chromatograms of blood and urine blanksamples and also model samples 1 processed according to the procedure 3 are given on Fig. 4.
Subjectively, the procedure 1 is characterized by the lowest level of co-extractive substances by the Fig. 2 of TLC-purification, but the procedure 3 has the worst results.
All procedures of sample preparation show the high efficiency of secnidazole isolation both for blood and urine (at the level of 90 % and higher). So high values of recovery give us possibility to calculate the main validation parameters without correction for the value of R. Calculation of linearity, accuracy and precision parameters has been carried out both with correction by the R value and without it -absence of such correction does not lead to significant worsening of the method validation parameters.      Tables 1-3   Table 1 The total results of recovery determination for HPLC/UV-procedures of secnidazole determination in blood and urine  Fig. 2. The chromatograms of blood and urine blank-and model samples: a -procedure 3, blood, blank-sample; b -procedure 3, urine, blank-sample; c -procedure 3, blood, model sample 1; d -procedure 3, urine, model sample 1 Table 2 The total results of linearity verification for HPLC/UV-procedures of secnidazole determination in blood and urine Accuracy and precision have been determined as within-run and between-run parameters, and we may not highlight the best procedure by the values of systematic and random errors, but according to the number of steps in the procedure of sample preparation the procedure 3 is optimal. a b c d e f Fig. 5. The calibration curves for HPLC/UV-procedures of secnidazole determination in blood and urine: a -procedure 1, blood; b -procedure 1, urine; c -procedure 2, blood; d -procedure 2, urine; e -procedure 3, blood; f -procedure 3, urine

Conclusions from the conducted research and prospects for further development of this field
The set of HPLC-procedures of secnidazole quantitative determination in blood and urine has been developed.
1. Three types of sample preparation have been proposed: 1) liquid-liquid extraction with organic solvents immiscible with water, 2) amphiphylic solvents extraction and salting-out with ammonium sulphate, 3) complex application of liquid-liquid extraction with organic solvents immiscible with water and amphiphylic solvents extraction with salting-out. All procedures are effective by the recovery parameter, but the second way of extraction is optimal.
2. Validation of the developed procedures has been carried out and it has been set that isopropanol application in the acid medium (рН = 2) is optimal for secnidazole determination in biological liquids -the extraction efficiency is maximal and equal to ∼97 %, and parameters of linearity, accuracy and precision are optimal.