DETERMINATION OF VALIDATION CHARACTERISTICS OF THE UV-SPECTROPHOTOMETRIC METHOD OF DOXYLAMINE QUANTITATIVE DETERMINATION IN BLOOD IN THE VARIANT OF THE METHOD OF STANDARD

With the purpose of improvement of carrying out the quantitative determinations in forensic and toxicological analysis the possibility of application of the method of standard for UV-spectrophotometric determination of analytes in biological fluids has been studied. The procedure for determination and acceptability estimation of linearity, accuracy and precision for validation of such methods in the variant of the method of standard tested by the example of UV-spectrophotometric method of doxylamine quantitative determination in blood has been offered. The given procedure provides application of the normalized coordinates. For normalization of the experimental data obtained two approaches have been used: 1) Approach 1: the use of the reference solution with the concentration of the analyte corresponding to its concentration in the final spectrophotometric solution measured under the condition of zero losses for the point of 100% in the normalized coordinates. 2) Approach 2: the use of the reference sample with the concentration of doxylamine corresponding to its concentration for the point of 100% in the normalized coordinates. Estimation of linearity, accuracy and repeatability of the method at the first stage has been performed using the model solutions within the approach based on assumption of insignificance of the uncertainty of the analyte quantitative determination in model solutions in comparison with the complete uncertainty of the analysis results ΔAs. At the second stage determination of linearity, accuracy, repeatability and intermediate precision of the method has been carried out on the model samples prepared using the matrix for three parallel runs. It has been shown that the method of standard can be applied for UV-spectrophotometric determination of doxylamine in blood – Approach 1 is more acceptable.

With the purpose of improvement of carrying out the quantitative determinations in forensic and toxicological analysis the possibility of application of the method of standard for UV-spectrophotometric determination of analytes in biological fluids has been studied.The procedure for determination and acceptability estimation of linearity, accuracy and precision for validation of such methods in the variant of the method of standard tested by the example of UV-spectrophotometric method of doxylamine quantitative determination in blood has been offered.The given procedure provides application of the normalized coordinates.For normalization of the experimental data obtained two approaches have been used: 1) Approach 1: the use of the reference solution with the concentration of the analyte corresponding to its concentration in the final spectrophotometric solution measured under the condition of zero losses for the point of 100% in the normalized coordinates.2) Approach 2: the use of the reference sample with the concentration of doxylamine corresponding to its concentration for the point of 100% in the normalized coordinates.Estimation of linearity, accuracy and repeatability of the method at the first stage has been performed using the model solutions within the approach based on assumption of insignificance of the uncertainty of the analyte quantitative determination in model solutions in comparison with the complete uncertainty of the analysis results Δ As .At the second stage determination of linearity, accuracy, repeatability and intermediate precision of the method has been carried out on the model samples prepared using the matrix for three parallel runs.It has been shown that the method of standard can be applied for UV-spectrophotometric determination of doxylamine in blood -Approach 1 is more acceptable.
Currently there is a number of international regulations given directive recommendations on carrying out the validation of bioanalytical methods -"Guidance for Industry: Bioanalytical method validation" (U.S. FDA, 2001) [5], "Guideline on validation of bioanalytical methods" (ЕМА, 2011) [7], "Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Specimens" (UNODC, 2009) [6] and "Standard Practices for Method Validation in Forensic Toxicology" (SWGTOX, 2012) [8].
The papers mentioned are oriented to development of methods for quantitative determination of analytes in biological fluids in the variant of the method of the calibration curve.The method of the calibration curve, undoubtedly, allows to take into account and partially level the influence of the matrix background absorption on the results of determination, but proves its value only in the case of performing routine analyses.In forensic and toxicological analysis we often meet with one-time examinations, and various biological fluids, organs and tissues are sent for the examinations, i.e. it is necessary to determine the analyte quantitatively in several various biological objects, but the necessity of carrying out such determinations can arise rarely enough.In such situation plotting the calibration curve for each matrix demands rather nonrational time consumption, and to the moment of obtaining the results of analysis they can become irrelevant.
Thus, it is of interest to study the possibility of using the method of standard when carrying out UV-spectrophotometric determination of analytes in biological fluids; in this connection the procedure for determination and acceptability estimation of linearity, accuracy and precision for validation of such methods in the variant of the method of standard [3] developed according to [1] has been offered.The results of testing of the approaches offered by the example of the UV-spectrophotometric method of doxylamine quantitative determination in blood are given in this paper [2].
The reference solution: place 400.0 mg of doxylamine succinate in a 100.0 ml volumetric flask, dissolve in the 0.1 mole/l of hydrochloric acid solution and dilute to the volume with the same solvent (standard solution 3, the concentration is 4000 mcg/ml).In a 100.0 ml volumetric flask place 18.00 ml of doxylamine succinate standard solution 3 using a burette and dilute to the volume with 0.1 mole/l of hydrochloric acid solution (standard solution 4, the concentration is 720 mcg/ml).In a 50.0 ml volumetric flask place 2.00 ml of doxylamine succinate standard solution 4 and dilute to the volume with 0.1 mole/l of hydrochloric acid solution (the reference solution, the concentration is 28.8 mcg/ml).
The model samples: 3 lines in 7 samples (20.00 ml) of the model blood (matrix) obtained from three different sources spiked with 1.00 ml of the process solutions 1-7, respectively.
The reference sample: a sample (20.00 ml) of the model blood spiked with 1.00 ml of the process solutions 8.
The solutions to be analysed: the solutions obtained by the validated method [2] for the model and reference samples.
The absorbance of the solutions to be analysed, model solutions and the reference solution was measured 3 times with taking out the cell at the wavelength of 262 nm by a SF-46 spectrophotometer in the cell with the layer thickness of 10 mm.As a compensation solution 0.1 mole/l hydrochloric acid solution was used.

Results and Discussion
Determination of validation characteristics of the UV-spectrophotometric method of doxylamine quantitative determination in blood was carried out according to the following procedure [3]: 1) the use of the normalized coordinates: (1) for normalization of the experimental data obtained two approaches were used: • Approach 1: the use of the reference solution with the concentration of doxylamine (С reference ) corresponding to its concentration in the final spectrophotometric solution measured under the condition of zero losses for the point of 100% in the normalized coordinates: (2) • Approach 2: the use of the reference sample with the concentration of doxylamine (С reference sample ) corresponding to its concentration for the point of 100% in the normalized coordinates: 2) the application range is 25 -125%, 25 -150%, 25 -175%; the mean lethal doxylamine concentration in blood [4] -25 mg/l (that corresponds to 36 mg/l of doxylamine succinate) was accepted as 100%; the number of concentration levels is g = 5, 6 or 7 (depending on the application range chosen) in constant increments of 25%; 3) estimation of linearity, accuracy and repeatability of the method at the first stage was performed using the model solutions within the approach based on assump-    tion of insignificance of the uncertainty of the analyte quantitative determination in model solutions in comparison with the complete uncertainty of the analysis results Δ As , according to the acceptability criteria calculated for the residual standard deviation , absolute term a model and correlation coefficient for the variants of the method application range offered, as well as to the requirements to accuracy and repeatability (δ model ≤ 2.05% and ≤ 6.40%); 4) at the second stage determination of linearity, accuracy, repeatability and intermediate precision of the method was performed on the model samples prepared using the matrix for three parallel runs; within each run the values of the linearity parameters, values δ and Δ Z were determined and compared with the calculated critical values -maxRSD 0 , maxа, minR с , maxδ and maxΔ Z ; for verification of intermediate precision the pooled mean value Z intra , the pooled relative standard deviation and the relative confidence interval were calculated for three runs obtained; the value should not exceed the extreme uncertainty of analysis maxΔ As : (4) The results of linearity, accuracy and repeatability determination of the UV-spectrophotometric method of doxylamine quantitative determination using the model solutions are given in Tab. 1 and 2.
The results of linearity, accuracy, repeatability and intermediate precision determination of the UV-spectrophotometric method of doxylamine quantitative determination using the model samples are given in Tab. 3, 4 and 5.
The data from Tab. 1-5 are the evidence that the method of standard can be applied for UV-spectrophotometric determination of doxylamine in blood; thus, the requirements offered to the validation parameters are completely performed only in the case of using Approach 1.

CONCLUSIONS
The procedure of determination and acceptability estimation of linearity, accuracy and precision offered previously [3] for validation of UV-spectrophotometric methods of quantitative determination of analytes in biological fluids used in forensic and toxicological analysis in the variant of the method of standard has been tested by the example of the UV-spectrophotometric method of doxylamine quantitative determination in blood.It has been shown that Approach 1 based on the use of the reference sample with the concentration of doxylamine corresponding to its concentration for the point of 100% in the normalized coordinates for normalization of absorbance values is more acceptable.Укр. журн. клін. та лаборатор. медицини. -2013. -Т. 8, №4. -С. 191-199. 3. Клименко Л.Ю. // Фармация Казахстана. -2014. -№4. -С. 42-48. 4

Table 1
Metrological characteristics of calibration straight lines Y = b•X + a obtained using model solutions of doxylamine succinate

Table 2
Results of accuracy and repeatability determination for the UV-spectrophotometric method of doxylamine succinate quantitative determination by model solutions

Table 3
Metrological characteristics of calibration straight lines Y = b•X + a for the UV-spectrophotometric method of doxylamine quantitative determination in blood without preliminary TLC-purification

Table 4
Results of accuracy and repeatability determination for UV-spectrophotometric method of doxylamine quantitative determination in blood without preliminary TLC-purification Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Specimens / United Nations Office onDrugs and Crime, Laboratory  and Scientific Section.-New York: United Nations, 2009.-70 p.

Table 5
Results of intermediate precision determination of the UV-spectrophotometric method of doxylaminequantitative determination in blood without preliminary TLC-purification