Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 2 of 14

 

Сontent

№	Description	Page
1.	The general outlines	3
1.1	The purpose of the work	3
1.2.	Material and equipment	3
1.3.	Description	4
1.4.	Method of conducting analysis on ФСП 42-53880 (ЛСР-007439/09-240909)	4
1.5.	Preparation of standard and analyzable solutions.	5
2.	Parameters of the analytical method.	7
2.1	Linearity	7
2.2	Accuracy	9
2.3	Convergence	11
2.4	Intra-laboratorial reproducibility	12
3.	Conclusion	13
4.	Literature	14

 

Process	Position	Surname, Name and Second Name.
Conduction of validation tests	Chemist AL Chemist AL	Samkova O.A. Zalyalova N.V.
Drawing-up of the validation report	Chemist AL Senior chemist AL	Samkova O.A. Nozhevnikova E.V.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 3 of 14

 

1. The general outlines.

1.1  The purpose of the work

Basing upon laboratory research, to confirm that the method of definition of ethylmethylhydroxipyrides succinate’s quantitative content in the medical agent  “Neurox, solution for intravenous and intramuscular injection 50mg/ml” with the help of UF-spectrophotometry  allows the generation of  reproducible and reliable result.

1.2  Material and equipment

Equipment:

Name	Model/version	Manufacturer	Serial Number	Verification Certificate	Valid till
Spectrophotometer	CARY-50	Varian	EL 04083277	034418	17.05.11
Software	CaryWinUV 3.00 (182)	Varian	-	-	-
Analytical Balance	XS-105 DU	MettlerToledo	1125422785	023869	08.12.10

 

Accessory equipment:

Name	Brand	State Standard Certification
Mohr-pipettes	Class 1;1 ml, 2 ml	State Standard Certification 20292
The volumetric dark-glass flasks	Class A; 1000, 100, 50 ml	State Standard Certification 1770

 

 

1.3  Description

   Results on the following parameters were achieved and processed in order to fulfill the task set:

• linearity;


• correctness; 


• intra-laboratorial reproducibility;


• linearity range

Closed joint-stock 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 4 of 14

 

   1.4. Method of conducting analysis on ФСП 42-53880 (ЛСР-007439/09-240909)

   Quantitative definition. Definition is conducted through spectrophotometric method. 

   2 ml of the drug are placed into a volumetric flask of 100 ml capaciousness, 0.01 Mvolume of solution is lapped with hydrochloric acid’s solution to the marker and stirred (as the solution under test).

   Optical density of the solution achieved is measured with spectrophotometer in a maximum absorption by 297nm length of the wave in the ditch of 10mm layer thickness. A 0.01M solution of hydrochloric acid is used thereby as a solution.

   Optical density of ethylmethylhydroxipyrides succinate is measured simultaneously. 

   The content of ethylmethylhydroxipyrides’ succinate in 1ml of the drug is calculated in grams (X) through a formula:

     

       D – stands for the optical density of the solution under test

         ‒ stands for the optical density of CO methylethylhydroxipyrides succinate

       a    ‒ the joint hinge of CO methylethylhydroxipyrides succinate.

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 5 of 14

 

100, 100 – dilution of the solution under test;

2 , 1 – quantity of the drug in ml, used in solutions;

P – content of the ground substance in CO ethylmethylhydroxipyrides succinate in percent.

Content of ethylmethylhydroxipyrides succinate in 1 ml of the drug must make up from 0.045 to 0.055g.

   Note 1. Preparation of CO ethylmethylhydroxipyrides’ solution.  About 0.1 g (exact band-and-hook hinge) of ethylmethylhydroxipyrides’ succinate (FSP-42-8455-07) is placed into a volumetric flusk of 100ml capaciousness, 50ml of 0.01M hydrochloric acid is added, and after absolute dissolution of the drug it is lapped with the solution with the same solvent to the marker and stirred.

    1ml of the solution achieved is placed into a volumetric flusk of 100ml capaciousness, the dissolvent is lapped to the marker and stirred.

   1ml of CO solution contains about 0.00001g ethylmethylhydroxipyrides succinate.

   Solution is exploited freshly prepared.

  

   1.5 Preparation of standard and analyzable solutions.

   1.5.1. Preparation of0.01 MHCl solution.

   100ml of 0.1M HCl solution is placed into a volumetric flask of 1000ml capaciousness. 300ml of water is added, stirred till complete dissolution, solution’s volume is lapped with the same solvent to the marker and stirred.

   1.5.2 Preparation of PCO ethylmethylhydroxipyrides’ succinate

   Standard sample

  

Name of the PCO	Line	Producer	P (content of the ground substance, %)
Ethylmethylhydroxipyrides’  succinate	020307	Limited Liability Corporation (LLC) “Bion”	99.4

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 6 of 14

  

succinate

“Bion”

      1. 0.06050g of ethylmethylhydroxipyrides’ succinate is placed into a volumetric flask of 100ml capaciousness, 50ml of 0.01M of hydrochloric acid’ solution is added and after complete dissolution of the drug, the solutions volume is lapped with the same solvent to the marker, and stirred. The concentration of ethylmethylhydroxipyrides’ succinate in the solution achieved makes up 0.03007 g/ml.

      2. 0.08021g of ethylmethylhydroxipirides’ succinate is placed into a volumetric flask of 100ml capaciousness, 50ml of 0.01M hydrochloric acid’s solution is added and after complete dissolution of the drug the solution’s volume is lapped with the same solvent to the marker, stirred. Concentration of ethylmethylhydroxipyrides’ succinate in the solution achieved makes up 0.03986 g/ml.

      3. 0.10298g of ethylmethylhydroxipyrides’ succinate is placed into a volumetric flask of 100ml capaciousness, 50ml of 0.01M hydrochloric acid’s solution is added and after complete dissolution of the drug the volume is lapped to the marker with the same solvent, and stirred. 1ml of the solution achieved is placed into a volumetric flask of 100ml capaciousness, solution’s volume is lapped to the marker, and stirred. Concentration of ethylmethylhydroxipyrides in the solution achieved makes up 0.05118 g/ml

      4. 0.12191g of ethylmethylhydroxipyrides’ succinate is placed into a volumetric flask of 100ml capaciousness, 50ml of 0.01M hydrochloric acid’s solution is added and after complete dissolution of the drug the volume is lapped with the same solvent to the marker, stirred. 1ml of the solution achieved is placed into a volumetric flask of 100ml capaciousness, solution’s volume is lapped to the marker, stirred. Concentration ethylmethylhydroxipyrides’ succinate in the solution achieved makes up 0.06059 g/ml.

     5. 0.l4316g of ethylmethylhydroxipyrides’ succinate is placed into a volumetric flask of 100ml capaciousness, 50ml of 0.01M hydrochloric acid’s solution is added and after complete dissolution of the drug, the volume is lapped with the same solvent to the marker, stirred. 1ml of the solution achieved is placed into a volumetric flask of 100ml capaciousness,

  

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 7 of 14

 

  the solvent’s volume is lapped to the marker, stirred. Concentration of ethylmethylhydroxipyrides’ succinate in the solution achieved makes up 0.07115g/ml.

   2. Parameters of the analytical method.

   2.1 Linearity

   Purpose: is to demonstrate, that the test’s results after mathematical processing are proportional to the concentration of the substance analyzed in the sample within the range of the interval given.

   Acceptability criterion: the criteria of linear dependence between concentration and optical density of the solution under analysis will be the correlation coefficient R higher than 0.99.

   The procedure of analysis

   Examination of the PCO ethylmethylhydroxipyrides’ succinate’s concentration linearity

   Five solutions of a standard working example with concentrations within the range between 60% and 140% of the quantity declared in the medicine “Neurox, solution for intravenous and intramuscular injection 50mg/ml”.

   The optical density of solutions obtained concerning the hydrochloric acid 0.01M in absorption maximum by the wave’s length of 297nm.

   The optical density’s measurements of the solutions obtained were conducted.

    The results obtained are represented in the table 1

    Table 1

№	Solutions optical density
	0.03007 g/ml	0.03986 g/ml	0.05118 g/ml	0.06059 g/ml	0.07115 g/ml
1	0.2322	0.3048	0.3865	0.4554	0.5316
2	0.2321	0.3048	0.3865	0.4555	0.5315
3	0.2321	0.3048	0.3863	0.4554	0.5317
n	3	3	3	3	3
average	0.2321	0.3048	0.3864	0.4555	0.5316
Standard deviation	0.000152	0.000153	0.00115	0.0001	0.00015
RSD	0.065	0.050	0.298	0.022	0.028

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 8 of 14

 

By the values of results achieved, a dependence of RSO ethylmethylhydroxipyrides succinate solution’s optical density on the concentration of ethylmethylhydroxipyrides succinate in the solution.

   Dependence of optical density on ethylmethylhydroxipyrides succinate’s concentration

  

 

   Y = 7.2859X + 0.0136

   The correlation coefficient has been calculated for valuation of linearity: R^2=1

   Conclusion:

1. It is demonstrated that the dependence of optical density of RSO ethylmethylhydroxipyrides succinate’s  solution on concentration of ethylmethylhydroxipyrides’ succinate in the solution within the limits of range from 60 to 140% of ethylmethylhydroxipyrides succinate’s quantity of the quantity declared in the medicine is linear.


2. The calculated coefficient of correlation R=1 meets the requirements of the acceptance criteria formulated and corroborates the linear dependence.


3. The method of quantitative definition proposed gives the linear dependence of PCO ethylmethylhydroxipyrides succinate solution’s optical density on concentration of ethylmethylhydroxipyrides’ succinate in the solution.

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 9 of 14

 

   2.2 Accuracy (correctness of the analytical method).

   Purpose: to define the accuracy (proximity) of the results achieved during the experiment to the true value using the solutions with the known concentration of ethylmethylhydroxipyrides succinate’s standard sample.

   Admissibility criterion:

1)     ratio error of definition must not exceed 2%.

2)     PCO’s true value must lie within the confidence interval’s limits, defined for the mean value calculated.

3)     Systematic error must miss.

   The Procedure of Analysis: for definition of the UF-spectrophotometry method’s accuracy, solutions, used for building gage curve, were analyzed. The mean value of optical density and ethylmethylhydroxipyrides succinate’s concentration, ratio error of definition were calculated for each solution according to three values.

   The results are represented in the table 2

 

Table 2

Parameter	Dimensionality	Level
		1	2	3	4	5
Valid concentration	mg/ml	0.03007	0.03986	0.05118	0.06059	0.07115
Defined concentration (mean value from 3 definitions)	mg/ml	0.02891	0.03902	0.05109	0.06051	0.07120
Ratio error	%	3.8	2.1	0.2	0.13	0.07
Mean value	%	1.26

 

   Define whether the mean value of RSO lies within the limits of confidence interval (define it for each RSO).

  The results are represented in the table 3

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 10 of 14

 

Table 3

Parameter	Dimensionability	Level
		1	2	3	4	5
Valid concentration (X true)	g/ml	0.03007	0.03986	0.05118	0.06059	0.07115
Defined concentration (X average)	g/ml	0.02999	0.03972	0.05109	0.06051	0.07120
Halfwidth of confidential interval	g/ml	0.001	0.001	0.001	0.001	0.001

   Define the presence of systematic mistake, therefore determine, whether the divergence between X average and x true is significant or not. Conduct the valuation using Student’s t-criterion for each RSO.

  

Parameter	Dimensionability	Level
		1	2	3	4	5
Valid concentration (X true)	g/ml	0.03007	0.03986	0.05118	0.06059	0.07115
Defined concentration (X average)	g/ml	0.02999	0.03972	0.05109	0.06051	0.07120
Standard dilatation	-	0.00152	0.00015	0.00011	0.001	0.00015
Student’s t-criterion calculated		0.3	1.6	1.4	1.3	0.3
Student’s t-criterion  tabular by P=0.99	4.03

   

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 10 of 14

Conclusion: t calculated<t tabular, thence there’s no statistically significant distinction, that is, the statistical error does not exist.

      2.3. Convergence

   Purpose: to demonstrate the convergence of results achieved by one chemist of the analytical laboratory OKK, achieved with the same equipment during short period of time.

   Admissibility criterion: in accordance with demands of analytical laboratory’s quality control board of CJSC “PharmFirm “Sotex””, relative standard departure (RSD) of results achieved must not exceed 2.0%.

   Prepare 6 samples of the solution under test. Determine the concentration of ethylmethylhydroxipyrides succinate within them, calculate RSD.

   The analysis procedure:

   2ml of the medical agent “Neurox, solution for intravenous and intramuscular injection 50mg/ml” is placed into a volumetric flask of 100ml capaciousness, 0.01M solution’s volume is lapped with HCl solution and stirred. 1ml of the solution achieved is placed inside volumetric flask of 100ml capaciousness, 0.01M HCl solution’s volume is lapped to the marker and stirred.

    6 samples are prepared in a way indicated above.

    The results achieved are represented in the table 5

    Table 5

Sample’s number	1	2	3	4	5	6
Optical density	0.3996	0.3835	0.3927	0.3904	0.3860	0.3882
Concentration g/ml	0.05066	0.05077	0.05202	0.05171	0.05110	0.05144
Mean value	0.051
Standard deviation, %	0.00053571
Relative standard deviation, %	1.04

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 12 of 14

 

   Conclusion:

   Relative standard dilatation(RSD) of parallel definitions – comprises 1.04%, which meets the requirement of admissibility criteria set and confirms the convergence of the results achieved. The results achieved are statistically equivalent.

 

      2.4. The intra-laboratorial reproducibility

     Purpose: evaluation of the intra-laboratorial reproducibility by the analysis of the same sample of MA “Neurox, solution for intravenous and intramuscular injection 50mg/ml” conducted by different chemists at a different time with complete repetition of the sample’s preparation procedure and measurements fulfillment.

  

   Admissability criterion: relative standard dilatation(RSD) of the parallel definitions must not exceed 1.0%; ;

   .

   The procedure of analysis: reproducibility of the chromatographic method was studied by the results of the series analysis of the preparation: “Neurox, solution for intravenous and intramuscular injection 50mg/ml”, CZL sample, achieved by two chemists of the analytical laboratory at a different time on the same equipment. The results are represented in the table 6. 

 Table 6

Sample №	Chemist 1	Chemist 2
1	0.05298	0.05144
2	0.05077	0.05133
3	0.05202	0.05085
4	0.05171	0.05116
5	0.05110	0.05095
6	0.05141	0.05072
Average	0.05128	0.051075
Statistical dilatation	0.0005357	0.000257
RSD, %	1.04	0.5

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 13 of 14

 

    Assessment of the difference between standard deflections of two series of tests was conducted by Fisher’s F-criterion,

F ,

where and  – are standard deflections of the unitary dimensions.

   Fisher’s calculated criterion =  = 4.34

   Numeral value of Fisher’s criterion by the fiducial probabilityP=0.99 and the number of the degrees of freedom fl=5 =10.97

  =4.34< =10.97, which means that the chemists achieved reproducible results.

   The validity of average results (P=99%) had been determined by Student’s t-criterion:

    , where S=  

   S= = =0.01134

 = 0.018077

   Student’s criteria for fiducial probability P=99% and for the number of degrees of freedom f=  

   Consenquently, 18< =3.17

   Conclusion: The mean values achieved from the tests conducted by different chemists at different time are statistically equivalent and meet the laboratory’s reproducibility criteria.

 

 

   3   Conclusion

   The method of quantitative definition of the active substance in the medical agent “Neurox, solution for intravenous and intramuscular injection 50mg/ml” meets the demands set and is eligible for the use in the laboratory of closed joint-stock “PharmFirm “Sotex”” Quality Control Department.

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 14 of 14

 

Table 7

Summary table on the method’s validation results

№	Parameter	Criteria adopted	Result achieved	Correspondence to the criteria set
1.	Linearity (Within the range of 60-140%)	>0.99	1	Yes
2.	Convergence	RSD 2.0%	1.04	Yes
3.	Intra-laboratorial reproducibility			Yes
4.	Correctness (within the range of 80-120%)	Ratio error of the definition is no more than 2.0%	1.26%	Yes

 

          4. Literature

      4.1 ФСП 42-53880 (ЛСР-007439/09-240909) “Neurox, solution for intravenous and intramuscular injection 50mg/ml”.

      4.3 N.A. Apstein The assessment of validity (Validation) of HPLC methods in the pharmaceutical analysis (review), chemistry-pharmaceutical magazine. Volume 38, № 4, 2004.

     4.4 K. Derffel, Statistics in the analytical chemistry. – M., Мир, 1994.  

  

 

  

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 2 of 14

 

Сontent

№	Description	Page
1.	The general outlines	3
1.1	The purpose of the work	3
1.2.	Material and equipment	3
1.3.	Description	4
1.4.	Method of conducting analysis on ФСП 42-53880 (ЛСР-007439/09-240909)	4
1.5.	Preparation of standard and analyzable solutions.	5
2.	Parameters of the analytical method.	7
2.1	Linearity	7
2.2	Accuracy	9
2.3	Convergence	11
2.4	Intra-laboratorial reproducibility	12
3.	Conclusion	13
4.	Literature	14

 

Process	Position	Surname, Name and Second Name.
Conduction of validation tests	Chemist AL Chemist AL	Samkova O.A. Zalyalova N.V.
Drawing-up of the validation report	Chemist AL Senior chemist AL	Samkova O.A. Nozhevnikova E.V.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 3 of 14

 

1. The general outlines.

1.1  The purpose of the work

Basing upon laboratory research, to confirm that the method of definition of ethylmethylhydroxipyrides succinate’s quantitative content in the medical agent  “Neurox, solution for intravenous and intramuscular injection 50mg/ml” with the help of UF-spectrophotometry  allows the generation of  reproducible and reliable result.

1.2  Material and equipment

Equipment:

Name	Model/version	Manufacturer	Serial Number	Verification Certificate	Valid till
Spectrophotometer	CARY-50	Varian	EL 04083277	034418	17.05.11
Software	CaryWinUV 3.00 (182)	Varian	-	-	-
Analytical Balance	XS-105 DU	MettlerToledo	1125422785	023869	08.12.10

 

Accessory equipment:

Name	Brand	State Standard Certification
Mohr-pipettes	Class 1;1 ml, 2 ml	State Standard Certification 20292
The volumetric dark-glass flasks	Class A; 1000, 100, 50 ml	State Standard Certification 1770

 

 

1.3  Description

   Results on the following parameters were achieved and processed in order to fulfill the task set:

• linearity;


• correctness; 


• intra-laboratorial reproducibility;


• linearity range

Closed joint-stock 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 4 of 14

 

   1.4. Method of conducting analysis on ФСП 42-53880 (ЛСР-007439/09-240909)

   Quantitative definition. Definition is conducted through spectrophotometric method. 

   2 ml of the drug are placed into a volumetric flask of 100 ml capaciousness, 0.01 Mvolume of solution is lapped with hydrochloric acid’s solution to the marker and stirred (as the solution under test).

   Optical density of the solution achieved is measured with spectrophotometer in a maximum absorption by 297nm length of the wave in the ditch of 10mm layer thickness. A 0.01M solution of hydrochloric acid is used thereby as a solution.

   Optical density of ethylmethylhydroxipyrides succinate is measured simultaneously. 

   The content of ethylmethylhydroxipyrides’ succinate in 1ml of the drug is calculated in grams (X) through a formula:

     

       D – stands for the optical density of the solution under test

         ‒ stands for the optical density of CO methylethylhydroxipyrides succinate

       a    ‒ the joint hinge of CO methylethylhydroxipyrides succinate.

 

 

 

 

Closed joint-stock “SOTEX”	Quality Control Department Analytic laboratory
Validation return	Quantitative definition
Validation type	Validation of analytical method
Protocol code: В/О-АЛ-МА-УФ-05-01	Page 5 of 14

 

100, 100 – dilution of the solution under test;

2 , 1 – quantity of the drug in ml, used in solutions;

P – content of the ground substance in CO ethylmethylhydroxipyrides succinate in percent.

Content of ethylmethylhydroxipyrides succinate in 1 ml of the