Quantitative analysis of piperidin-1-ium {[5-(2-furyl)-4-phenyl-4 H -1,2,4-triazol-3-yl]thio}acetate, substance of the veterinary drug «Tryfuzol», in poultry meat by LC–DAD–MS

Tryfuzol, Triazoles, Poultry, Chromatography, High Pressure Liquid, ESI Mass Spectrometry. Aim. Development of sensitive, acurate, reproducible HPLC-DMD-MS method for determination of the residual amounts of active drug substance «Tryfuzol» in poultry homogenate samples and application of this method for testing chiken groups is an important task for the con ﬁ rmation of this drug safety. Methods and results. The substance extraction conditions were optimized for methods development. It was established optimum extraction condition. Chromatographic conditions and mass spectrometric studies were optimized. As mobile phase, a ﬂ ow rate of 0.4 ml min -1 isocratic elution 0.1% (v/v) formic acid in water (A) acetonitrile containing 0.1% (v/v) formic acid (B). The composition of the mobile phase of 70% / 30% (v/v). Total running time is 5 minutes. Wavelength of the detector is 276 nm. Control of substances a vailability was carried by retention time and under UV and mass spectra. Conclusion. The technique complies with relevant regulations. The absence of said substance in the study chiken group was proven by using developed techniques.


Instruments
The device is LC MS: Agilent 1260 Infi nity HPLC System (degasser, binary pump autosampler, single quadrupole mass spectrometer Agilent 6120 with ionization in electro-spray API-ES (ESI); OpenLAB CDS Software.

Chromatographic Conditions
An Agilent ZORBAX SB-C18 analytical column (30 mm x4.6 mm; 1.8 μm, Agilent Corporation) with guard column was used. The column temperature was set at 40 o C and the injection volume was 2 μL. The mobile phase, pumped at a fl ow rate of 0.4 mL min -1 with isocratic elution, consisted of 0.1% (v/v) formiate acid in water (A), acetonitrile containing 0.1% (v/v) of formic acid (B). The mobile phase composition was 70% A/30% B (v/v). The total run time was 5 min. DAD wavelength was 276 nm.

Mass-spectrometric conditions and confi rmation of tryfuzole and IS presence
Mass-spectrometer conditions was choosen to obtain maximal response: 1) scan mode for the identifi cation of the peak m/z from 250 to 310; 2) positive polarity; 3) the drying gas rate (nitrogen) -10 L/min; 4) capillari Voltage 4,000 V; 5)drying gas temperature, fragmentor voltage, nebulaizer preassure are presented at the Table 1. Optimization of the ion-source conditions was conducted by fl ow injection analysis (direct introduction of the sample into the ionization chamber without chromatografi c separation) by full factorial design. Statistical analysis of the results was performed on a personal computer employing a Statistica Package v. 8.0 (StatSoft, USA) based on the values of full factorial design and the corresponding peak areas. The polynomial regression equations was determined. The optimal values of factors was found according calculated equations using Solver (Optimization tool for Excel, Frontline Systems, Inc., USA).
The ions for the analytes were monitored by (m/z): tryfuzole 302.1, IS 256.0.

Preparation of Solutions of Standard Calibration and Quality Control Samples
Standard solutions of the tryfuzole substance (1 mg mL -1 ) were prepared by dissolving appropriate amounts of these reference substances in the mixtures of 0.1% HCOOH in CH 3 CN and 0.1% HCOOH in H 2 O -30:70, 40:60 and 50:50 respectively.
Standard solutions of the tryfuzol substance for the determination of the recovery at SPE (0.05 mg mL -1 ) were prepared by mixing of 50 μL of 1 mg mL -1 solutions of reference substance with appropriate amounts of the mixtures of 0.1% HCOOH in CH 3 CN and 0.1% HCOOH in H 2 O -30:70, 40:60 and 50:50 respectively.
Non-extracted reference tryfuzol substance and IS for determination of the recovery at full extraction (SPE and liquid extraction) (0.07 mg mL -1 ) were prepared by mixing of 70 μL of 1 mg mL -1 solutions of reference substances with appropriate amounts of the mixtures of 0.1% HCOOH in CH 3 CN and 0.1% HCOOH in H 2 O -30:70, 40:60 and 50:50 respectively.
Standard calibration solutions of the tryfuzol substance (1.2; 1.6; 2.0; 2.3; 2.5; 6.0; 14.3 mg mL -1 ) were prepared by the disolvation of the appropriate amount of the tryfuzole substance in the highly purifi ed water.
Series of the tryfuzol calibration solutions were made by addition of 1 ml standard solution to the 30 g of homogenate and mixed. Then they were extracted by the 40 mL mixure of 0.1% HCOOH in CH 3 CN and 0,1% HCOOH in H 2 O -40:60 (v/v) according to the procedure (Fig.2 and Fig. 3). B. O. Varynskyi, Ye. G. Knysh, V. V. Parchenko, O. I. Panasenko, A. G. Kaplaushenko Quality control solutions (QC) were made by the same way, by the addition of 1 ml standard solution to the 30 g of homogenate and mixed. Then they were extracted by the 40 mL mixure of 0.1% HCOOH in CH 3 CN and 0.1% HCOOH in H 2 O -40:60 (v/v) according to the procedure ( Fig.2 and Fig. 3). The fi nal tryfuzol concentration were at the lower limit of quantifi cation (LLOQ), within three times of the LLOQ (low QC), around 30-50% of the calibration curve range (medium QC), and at least at 75% of the upper calibration curve range (high QC). The fi nal content of the tryfuzol substance in the standard gomogenate were 40, 70, 195 and 390 μg mL -1 for trifuzole.  All standard solutions were stored at 5 o C, either for calibration curves of analyte or quality control (QC) in the pre-study validation and during the study.
Sample liquid extraction 30 gram of the meat was homogenized with a blender. 240 μL of the 1% IS was added and was mixed. After 2 hours 40 mL 40% CH 3 CN with 0.1% HCOOH was added and was mixed. After 2 hours extract was fi ltered through a glass fi lter. It was centrifugated at 15,000 g 10 min and was fi ltered through nylon syringe fi lter ID 13 mm, pore size 0.2 μm, (Fig.2).
Solid phase Extraction Procedure of the solid phase extraction was shown at the Fig. 3. SPE C18 cartridge (100 mg/1mL) was conditioned by the 1 mL CH 3 CN and 1 mL of the extract obtained according to the procedure (Fig.2

) was added into SPE cartridge
Validation of the Method The specifi city was confi rmed by analyzing blank samples to determine the absence of interference with analyte.
Analytical signal LLOQ sample: the analyte signal of the LLOQ sample should be at least 5 times the signal of a blank sample [1,2].
Within-run precision and accuracy of drug «Tryfuzole» substance determination were determined by QC samples analyzing at four different concentrations: at LLOQ, low (within three times the LLOQ), medium (around 30-50% of the calibration curve range) and high (at least at 75% of the upper calibration curve range) concentrations.

Application of the Analytical Method
Determination of residual amounts of drug substance conducted according to procedure (Fig. 2,3), comparing with homogenate samples with the addition of the standard solution of the drug «Tryfuzol» substance.

Calculation of concentration
The calibration graph equation was calculated by the method of external standard that should be checked every time during the research conditions.

LC-UV and MS Optimization
Earlier authors [1][2][3] described HPLC conditions of the determination number of derivatives of 1,2,4-thiotriazoles. The acetonitrile was applied as the organic modifi er. The authors suggested using acidic pH less than 3.0; while phosphoric acid chosen as acidifier. Low pH decrease ion-exchange mechanism of interaction of nitrogen-content bases with silanole groups and improve shape of the peaks, also increases retention due to interaction of protonated acidic molecules with reverse-phase sorbent [9][10][11]. We used prompted formiate acid as the more volatile and more convenient to use with mass spectrometer detector.
DAD detection wavelength was choosen according to the maximal adsorption and equial 276 nm. The ultraviolet spectra is showing at the fi g. 4,5. ES ionizatision is a soft method, so easy to receive unfragmented ions. Analytes and IS respond best to positive ionization, so protonated molecular ions [M+H] + were present as major peaks for compounds. The mass spectra of the protonated molecules by m/z from 250 to 310 are presented in Fig. 6,7. The presence of the molecules can be confi rmed by the correspondent UV and MS spectra.
The composition of the mobile phase is an important factor that affects the ESI processes. A high content of organic composition in the mobile phase to reduce signal suppression. But high percentage of acetonitrile dramatically decreases retention and also selectivity of determination. We chose 30% CH 3 CN. It was enough for signal intensity, selectivity and time of analysis was only 5 min in isocratic conditions. Solid Phase Extraction development Due to the high Log P of the tryfuzole acidic form equal 3.48±0.66 (it was calculated with ACD labs 6.0 Software), we decide that tryfuzol molecule should have good retention at non-polar phase, so we chose C18 SPE cartridge for the adequate retention. Maximal LogD exist in acidic medium from pH=2 to pH=3. So we chose 0.1% HCOOH as acidifi er (pH~2.7), Log D is equal to 3.34. Also extraction of the tryfuzole from meat we produced by mixture of acetonitrile-water with 0.1% HCOOH. 3 standard solutions (0.05 mg/mL) with different concentration of the acetonitrile: 30%, 40%, 50% with 0.1% HCOOH was prepared. We used water for the washing due to small elution of analyte. Acetonitrile used for the elution of analyte due to strong elution force. Maximal recovery was at 30% CH 3 CN ( Table 2). Fig. 4. UV spectrum of the tryfuzole.      Table 4 Accuracy and precision of the substance determination method of the drug «Tryfuzol» in homogenate (n = 5) by 4 concentration levels Fig. 8. LLOQ chromatograms of chiken meat spiked with two analytes and IS.

Sample Liquid Extraction development
Homogenized meat (100 g) was spiked by the 1 mL 1% substance of the drug «Tryfuzol», 1 mL 1% IS and mixed. It was devided into 3 samples about 30 g. The sample tryfuzole content ~3 mg. Each sample was extracted by the 45 mL acetonitrile-water mixture with 0.1 % HCOOH. Acetonitrile content was respectively 30%, 40%, 50%. Final concentration of tryfuzole and IS in the extacts was ~ 0.07 mg/mL.
We chose mixture of acetonitrile-water with 0.1% HCOOH as extragent of the tryfuzole from meat for the destruction of binding with proteins. We studied 30%, 40%, 50% acetonitrile with 0.1% HCOOH ( Table 3). Most recovery was at 40% CH 3 CN.

Selectivity and Specifi city
Diode-array detection at 276 nm was quite selective. The substance of the drug «Tryfuzol» and IS were chromatographically separated with the retention time of 3.4 and 1.4 respectively. Additionally, the selectivity of the method was provided by the mass spectra substances determination. Total chromatography time was 5 min. Interference with impurities is absent (Fig. 8).
Linearity of Calibration Curves and LLOQ Calibration curve was built on the basis of the depending diode-array detector response at a wavelength of 276 nm on the content of the substance in homogenate, that is performed by an external calibration standard. Calibration graph was linear for 40-475 μg/g substance in the homogenate. The satisfactory linearity was obtained. The corresponding equation was received: y=2,4198x-57,92; R²=0,997.

Assay Precision and Accuracy
The content of the drug substance «Tryfuzol» in the QC samples were determined using external standard calibration graph equation. Accuracy and precision was determined for the substance quality control solutions (QC). Accuracy and precision of data given in the Quantitative analysis of piperidin-1-ium { [5-(2-furyl)-4-phenyl-4H-1,2,4-triazol-3-yl]thio}acetate, substance of the veterinary drug «Tryfuzol»...

Application of the Analytical Method
This method was reproducible, accurate and sensitive, and can be used successfully for determination of the active ingredient of the drug «Tryfuzol» residual amounts in the poultry meat homogenate [7,8]. Samples of poultry (30 g) were treated in accordance with the method (Fig. 2 and Fig. 3) and compared with an extract made from the standard substance drug «Tryfuzol» sample (Fig. 9). Active substance of the drug «Tryfuzol» was not detected (Fig. 10).

Content Calculations
Due to the better reproducibility we used only external standard calibration equation for content calculations. Internal standard we used for the extraction conditions control.

Conclusions
1. The method capable for identifi cation «Tryfuzole» active substance residues at ≥18 μg/g and for the determination of «Tryfuzole» active substance residues in poultry meat homogenizate samples at ≥30 μg/g levels was elaborated.
Prospects for further research Elaborate methods of determination of the active substance of drug «Tryfuzol» in the blood plasma and other biological fl uids.