ANALYTICAL DIAGNOSING OF MILNACIPRAN POISONINGS

The method of milnacipran isolation with chloroform from the dehydrated biological material with subsequent extrac - tion purification in the n-hexane-acetonitrile solvent system has been developed. The method developed has allowed to isolate 47±5% of the antidepressant. The TLC-screening method of a number of antidepressants has been developed using four mobile phases with a low correlation and the sequential scheme of visualization by a set of chromogenic reagents. It has allowed to separate milnacipran, venlafaxine, amitriptyline, fluoxetine and sertraline. As differentiating reagents the Liebermann’s reagent and the Mandelin’s reagent in modification consisting in sequential treatment of the sample by the Mandelin’s reagent and formaldehyde vapours have been suggested. The methods of identification and quantitative determination of milnacipran in the biological material after the TLC purification using HPLC with multiwave UV-spectro photometric detection have been developed. The calibration curve of the dependence of the peak area on the concentration was described by the following equation: Y=5.14·10 -5 X; linearity was within the concentration range of 24.2-500 µg/ml; LOD and LOQ were 8.0 and 24.2 µg/ml (at 262 nm), respectively. The results obtained can be used in forensic toxicology for diagnosing milnacipran poisonings.

M іlnaсipran -(1R,2S)-rel-2-(Aminomethyl)-N,Ndiethyl-1-phe nyl cyclo pro panecarb oxami de is a novel third-generation thymoleptic. Its pharmacological effect is due to the dual action, it is a selective serotonin and norepinephrine reuptake inhibitor (SNRI). Mіlnaсipran is used for the treatment of moderate and severe endogenous depression [3]. Fatal intoxications associated with mіlnaсipran overdoses and co-administration of fluoxetine and sertraline in therapeutic doses [8], as well as ethanol [10] have been reported, in these cases peripheral blood concentrations of mіlnaсipran are 21.5 mg/l and 3.15 μg/ml, respectively.
Bioanalytical methods for analysis of mіlnaсipran in the blood and plasma using high pressure liquid chromatography with UV spectrophotometric [7], diode array [12] and MS- [5] detection have been developed. Methods of analysing the biological material for the presence of mіlnaсipran have not been developed. The general isolation methods appeared to be ineffective for a number of antidepressants, in particular for ami-triptyline, fluoxetine and sertraline [1,2], due to the lipophilic properties of these substances [6]. In this regard, the practical interest is the study of the efficiency of mіlnaсipran isolation (Vd=5.5 l/kg [6]) from the biological material with chloroform as a lipophilic solvent with subsequent extraction purification in the n-hexane-acetonitrile solvent system [4]. Therefore, the aim of this study was to determine the optimal conditions for mіlnaсipran isolation from the biological material, develop the TLCscreening scheme for the group of antidepressants, which can be co-administrated with mіlnaсipran, as well as the methods of identification and quantitative determination for mіlnaсipran in the biological material by HPLC with multiwave UV-spectrophotometric detection.

Materials and Methods
The method of mіlnaсipran isolation with chloroform with subsequent extraction purification in the n-hexane-acetonitrile solvent system. Add 1 ml of the aqueous solution containing 574 mg of mіlnaсipran hydrochloride (corres-ponding to 500 mg of the mіlnaсipran base) to the powdered biological sample (5 g of the liver tissue) and allow to stand for 24 h. Simultaneously perform the blank experiment.
Isolation of mіlnaсipran from the liver with chloroform was carried by the method given in the work [7].
The method of Thin Layer Chromatography. Two types of chromatographic plates -Merk (Silica gel 60 F254, 10x20 cm in size) and Sorbfil (PTLC-P-А) (10x10 cm in size) were used for thin-layer chromatographic studies.
Evaporate 10-30 ml aliquots of the final chloroform extract obtained from the tissue spiked with mіlnaсipran and extracts from the blank tissue (drug-free) to the minimum volume (~ 0.05 ml) and spot as a band onto the start line. Next to it spot 10 ml of the standard solution of mіlnaсipran in methanol (1 mg/ml). At first develop the chromatograms in chloroform to separate the drug from endogenous impurities, then use four mobile phases (listed below).
Thin-layer chromatographic studies were performed as described in the work [1,2,4]. The antidepressant was detected on the plate with the chromogenic reagents pre-S.V.Baiurka -Candidate of Pharmacy, associate professor of the Department of Toxicological Chemistry of the National University of Pharmacy (Kharkiv) sented in Tables 1, 2. Elute mіlnaсipran from the chromatogram band untreated by the location reagents with methanol. Evaporate the eluate, and reconstitute the residue 1 ml of methanol.
The method of HPLC. The HPLC study was performed using a "MiLi-Chrome A-02" microcolumn high pressure liquid chromatograph with a multiwave UV-spectrophometric detector by the method presented in the work [4]. The injected volume was 10 μl. Quantitative determination of mіlnaсipran in eluates from chromatograms was carried out at the wavelength of 262 nm.

Results and Discussion
According to the epidemiological studies [9] the most poisonings with novel antidepressants are combined. The scheme of mіl-naсipran detection by thin layer chromatography screening method in the presence of a number of antidepressants from different groups such as venlafaxine (SNRI), amitriptyline (TCA), sertraline and fluoxetine (SSRI) has been developed.
The simultaneous use of four mobile phases (MP) with a high distributive power in relation to drugs under study and the lowest correlation between them was proposed. They are ethyl acetatemethanol -25% ammonium hydroxide solution (85:10:5) (MP 1), methanol -25% ammonium hydroxide solution (100:1.5) (MP 2), cyclohexane -toluene -diethylamine (75:15:10) (MP 3), and toluene -acetone -ethanol -25% ammonium hydroxide solution (45:45:7.5:2.5) (MP 4). The Rf values of antidepressants in the mo-bile phases selected on two types of the chromatographic plates (Merk and Sorbfil) are shown in Table 3. The Dragendorff's reagent modified by Munier and acidified iodoplatinate solution were the most sensitive common reagents for detection of the substances studied. As differentiating reagents the Liebermann's reagent and the Mandelin's reagent in modification consisting in sequential treatment of the sample by the Mandelin's reagent and formaldehyde vapours were suggested (Table 1). Using a particular set of additional chromogenic reagents (Table 2) it was proposed to use four reagents, and it was sufficient for reliable identification of toxic substances according to the TIAFT recommendations [7] for the final stage of visualization. The degree of mіlnaсipran elution with methanol from Table 1 The  Table 4). The method showed linearity in the range of 24.2-500 mg/mL. The LOD and LOQ values were calculated based on the parameters of the calibration curve; they were 8.0 μg/ml and 24.2 μg/ml, respectively. Accuracy and precision of the method developed were 101.8 % (RSD= =1.4%) at the low concentration level, 100.8% and 100.5% (RSD= =1.0%) at the middle and high concentration levels, respectively. Therefore, they satisfy the requirements for the methods used in forensic toxicology [11].

results of antidepressant visualization in TLC-screening
The method of mіlnaсipran isolation with chloroform was quite efficient and allowed isolating 47±5% of the antidepressant studied ( Table 4). The results obtained can be used in forensic toxicology for diagnosing mіlnaсipran poisonings. CONCLUSIONS 1. The method of milnacipran isolation with chloroform from the biological material dehydrated by triturating with anhydrous sodium sulphate with subsequent extraction purification in the n-hexaneacetonitrile solvent system has been developed. The method developed has allowed to isolate 47±5% of the medicine.
2. The TLC-screening method of a number of antidepressants has been developed using four mobile phases with low correlation and the sequential scheme of visualization by chromogenic reagents. It has allowed to separate milnacipran, venlafaxine, amitriptyline, fluoxetine and sertraline.
3. The sensitive and specific methods of identification and quantitative determination of milnacipran in the biological material by HPLC with multiwave UV-spectrophotometric detection have been developed. Table 3 The Rf values of antidepressants in TLC-screening systems  Table 4 The results of HPLC determination of mіlnaсipran isolated from the liver with chloroform followed by purification in the n-hexane-acetonitrile system