Разработка и валидация методик спектрофотометрического определения каптоприла в крови по реакции с реактивом Эллмана

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Аналiз та стандартизацiя бiологiчно активних сполук та лiкарських форм
Analysis and standardization of biologically active substances and dosage forms
UDC 615. 225. 2:543. 422. 3/7:543. 054
L. Yu. Klimenko1, Z. V. Showkova1, O. Ye. Mykytenko1, A. G. Kaplaushenko2
Development and validation of the methods of captopril spectrophotometry determination in blood by the reaction with the Ellman reagent
1National University of Pharmacy, Kharkiv, 2Zaporizhzhya State Medical University
Aim. To rationalize quantitative determinations in forensic and toxicological analysis, the possibility to use the method of standard for analytes quantitative determination in biological fluids has been studied.
Methods and results. The series of spectrophotometric methods of captopril quantitative determination in blood based on reaction with the Ellman reagent has been developed using amphiphylic solvents (isopropanol, acetonitrile, methanol) in condition of aqueous phase saturation by ammonium sulphate. Validation of the developed methods in the variant of the method of standard has been carried out, conformity of the obtained values of validation parameters to the acceptability criteria has been shown.
Conclusion. It has been set that acetonitrile application in the acid medium (рН=2) is optimal.
Розробка та валвдащя методик спектрофотометричного визначення каптоприлу у KpoBi за реакщею
з реактивом Еллмана
Л. Ю. Клименко, З. В. Шовкова, О. С. Микитенко, А. Г. Каплаушенко
З метою рацюнатзаци здшснення кшьюсних визначень у судово-токсиколопчному ана^ вивчили можливгсть використання методу стандарту для кшьюсного визначення аналтв у бюлопчних рщинах. Розробили серш спектрофотометричних методик кшьюсного визначення каптоприлу у кров^ що Грунтуються на реакци з реактивом Еллмана, з використанням aмфiфiльних розчинниквзопропанолу, ацетоштрилу, метанолу) в умовах насичення водшл фази амошю сульфатом. Виконали вaлiдaцiю методик у вaрiaнтi методу стандарту, показали вщповщтсть значень вaлiдaцiйних пaрaметрiв, яю отримали, до критерш прийнятностi. Встановили, що оптимальним е використання ацетонприлу в кислому середовищi (рН = 2).
KrnKoei слова: валiдацiя, бiоаналiтичнi методики, каптоприл, реактив Еллмана, спектрофотометрiя.
Актуальт питання фармацевтичног i медичног науки та практики. — 2014. — № 3 (16). — С. 8−13
Key words: Validation Studies, Analitical Chemistry Techniques, Captopril, Ellman'-s reagent, Spectrophotometry.
Разработка и валидация методик спектрофотометрического определения каптоприла в крови по реакции
с реактивом Эллмана
Л. Ю. Клименко, З. В. Шовковая, Е. Е. Микитенко, А. Г. Каплаушенко
С целью рационализации проведения количественных определений в судебно-токсикологическом анализе изучена возможность использования метода стандарта в ходе количественного определения аналитов в биологических жидкостях. Разработана серия спектрофотометрических методик количественного определения каптоприла в крови, основанных на реакции с реактивом Эллмана, с использованием амфифильных растворителей (изопропанола, ацетонитрила, метанола) в условиях насыщения водной фазы аммония сульфатом. Проведена валидация разработанных методик в варианте метода стандарта, показано соответствие полученных значений валидационных параметров критериям приемлемости. Установлено, что оптимально использование ацетонитрила в кислой среде (рН=2).
Ключевые слова: валидация, биоаналитические методики, каптоприл, реактив Эллмана, спектрофотометрия.
Актуальные вопросы фармацевтической и медицинской науки и практики. — 2014. — № 3 (16). — С. 8−13
The necessity of analytical methods'- validation currently becomes the vital and widely discussed problem in analytical toxicology [1,2].
The available international guidance on carrying out validation of bioanalytical methods [3,4] are reckoned, firstly, on using exclusively chromatographic methods of analysis, and secondly, on application of the method of calibration curve that implies carrying out a lot of routine analyses in practical work. Single examinations are more widespread in practice of forensic and toxicological analysis, and application of the method of standard is more justified in this situation.
Purpose of the work:
• developing the series of methods of captopril quantitative determination in blood using different procedures of sample preparation based on spectrophotometric
methods offered before [5] by reaction with the Ellman reagent-
• carrying out validation of the offered methods and estimating the possibility of the method of standard application for captopril spectrophotometric determination in blood-
• choosing the optimal procedure of sample preparation which provides effective captopril isolation from blood and low content of co-extracted substances in the obtained extracts at the minimum value of the method.
Materials and methods
Captopril of pharmacopoeia purity was used in the experiment. The order of preparation of standard, process and model solutions, and also model samples are presented onfig. 1.
standard solution 1
ms= 1000.0 mg Vm. f= 200.0 ml H2O
1
5000 mcg/ml
'- standard solution 2
m5 = 50.0 nig VW*=500.0 ml phosphate buffer solution with pH — 8
. 100 mcg/mi
process solutions 1−7
Vi = 28. 00- 24. 00- 20. 00- 16. 00- 12. 00- 8. 00- 4. 00 ml VW= 100,0 ml H2O
I
1400- 1200- 1000- 800- 600- 400- 200 mcg/ml
^ reference % solution
V2 = 16. 00 ml Ут. 1−100.0 ml phosphate buffer solution with pH = 8
¦ standard ¦ solution 3
ms = 50.0 mg Vm. f= 500.0 ml phosphate buffer solution with pH = 8 I
100 mcg/ml ,
model samples
(3 sef from 3 sources) 1. 00 ml VW* = 20. 00 ml I
C*& quot-** = 70- 60- 50−40- 30- 20- 10 mcg/ml
model solutions 1 — 7
V3 = 28. 00- 24. 00- 20. 00- 16,00- 12. 00- 8. 00- 4. 00 ml IV, = 100.0 ml phosphate buffer solution with pH = 8 1
v C™*'- = 28- 24- 20- 16- 12- 8−4 mcg/m! y
Fig. 1. The procedure of solutions and samples preparation for validation of spectrophotometric methods of captopril determination in blood.
Fig. 2. The main stages of the methods of captopril spectrophotometric determination in blood.
The design of experiment on development of methods of captopril spectrophotometric determination in blood by reaction with the Ellman reagent is presented on fig. 2.
The procedure of the Ellman reagent preparation: 50.0 mg of 5,5'--dithio-bis (2-nitrobenzoic acid) were placed in the measuring flask with the capacity of 100.0 ml, a little amount of the phosphate buffer solution with pH = 6.8 and 2.5 ml of the 0,1 mole/l sodium hydroxide solution were added to complete dissolution of 5,5'--dithio-bis (2-nitroben-zoic acid), the solution was diluted to the volume with the phosphate buffer solution with pH = 6.8. The Ellman reagent pH was controlled with the help of ionometer pH-150 (the pH value of the solution should not exceed 7. 0).
The model (see fig 1) and blank-samples were analysed for each developed method- the blank-samples were prepared in the following way: 1) 5 samples (20. 00 ml) of the blood
obtained from the different sources, 1. 00 ml of distilled water were added into them- 2) 3 samples (20. 00 ml) of distilled water.
The absorbance of the solutions was measured 3 times with randomization of cell position.
Results and discussion
The approaches to validation of quantitative determination methods for forensic and toxicological analysis as to the instrument of optimal method development within the assigned purpose have been offered by us before [6 — 8] - the procedures of experiment carrying out properly and acceptability criteria of the obtained results have been developed in the variant of the method of standard.
For captopril determination the spectrophotometric method based on the photometric reaction with the Ellman reagent at pH = 8 [5] has been developed by us before and the procedure
of mentioned analyte isolation from blood by maceration with the 1 mole/l hydrochloric acid solution and subsequent extraction with the mixture of chloroform and isopropanol (8: 2) in the acid medium (pH = 2) [9] - the efficiency of captopril isolation from blood using this procedure has been evaluated by means of the spectrophotometric method mentioned above and is equal to ~75%.
In the present work it has been suggested to carry out capto-pril isolation from blood by its maceration with amphiphylic solvents and subsequent separation of organic layer under the conditions of aqueous phase saturation by electrolyte for increasing the efficiency- this approach enjoys wide popularity in modern forensic and toxicological analysis [2, 10]. Such amphiphylic solvents as methanol, isopropanol and acetonitrile were used in the experiment- ammonium sulphate was applied as electrolyte for saturation of aqueous phase. Isolation was carried out in the acid medium (pH = 2) — as in the method offered before [9].
Thus, the development of the series of methods of captopril determination in blood using the method of spectrophotome-try by reaction with the Ellman reagent has been become the result of this stage of investigations- the methods differ by the procedures of sample preparation (fig. 2).
For choosing the optimal methods of captopril determination in blood we carried out their validation 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 [6−8].
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 absorbance of 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 absorbance values of the model samples.
The range of the methods application is D = 25 — 175%- the number of concentration levels is g = 7 in constant increments of 25%- as 100% the mean toxic captopril concentration in blood [2] - 40 mcg/ml — is accepted.
The methods validation was carried out at the first stage using model solutions (fig. 3) and proceeding from two approaches [6]:
Approach 1: the uncertainty of analyte quantitative determination in model solutions A^ is insignificant against the total uncertainty of analysis results.
Approach 2: the uncertainty of sample preparation procedure is equal to the uncertainty of analyte quantitative determination in model solutions AT& quot-1'-.
Fig. 3. The stages of validation of spectrophotometric method of captopril determination using model solutions.
The total results of validation are presented in table 1 and allow to point to the conclusion about acceptable linearity, accuracy and repeatability of the method of captopril quantitative determination by the method of spectrophotometry by reaction with the Ellman reagent in the variant of the method of standard- at the same time it is necessary to note that the requirements to accuracy are fulfilled only within Approach 2.
Thus, it is possible to recommend the method of captopril quantitative determination by the method of spectrophotometry by reaction with the Ellman reagent to further application in forensic toxicology with the purpose of development of the methods of biological objects analysis for captopril quantification.
The total results of validation of spectrophotometric method of captopril determination by reaction with the Ellman reagent, which were obtained using model solutions
Table 1
linearity Parameter
bmodel model sb a^odel S^odel a RSD0model RSDmodel c
1. 019 0. 011 0. 130 1. 257 1. 487 0,9997
acceptability criterion Approach 1 — - amodel & lt- 2. 73% amodel & lt- 2 015 ¦ smodel a & lt- 3,18% & gt- 0,9983
satisfied satisfied satisfied satisfied
Approach 2 — - amodel & lt- 6. 03% amodel & lt- 2 015 ¦ smodel a & lt- 7,02% & gt- 0,9915
satisfied satisfied satisfied satisfied
accuracy and repeatability Parameter
Zmodel RSDmodel z Qmodel Amodel z
102. 54 2. 24 2. 54 4. 35
acceptability criterion Approach 1 — - & lt- 2. 05% & lt- 6. 40%
unsatisfied satisfied
Approach 2 — - & lt- 4. 52% & lt- 14. 14%
satisfied satisfied
Table 2
The total results of validation of spectrophotometric methods of captopril determination in blood by reaction with the Ellman reagent
Parameter Solvent acceptability criterion
(CH3)2CHOH CH3CN CH3OH
specificity
0. 019 0. 020 0. 024 —
RSD (blank) 5. 41 4. 87 5. 73 & lt- 6. 71%
5b|ank (25%/50%) 9. 80 / 5. 23 9. 64 / 5. 12 12. 33 / 6. 73 & lt- 8. 00% / 6. 40%
0. 005 0. 005 0. 006 —
2. 70 2. 39 3. 14 & lt- 0. 32 бы k
recovery
R 90. 40 96. 80 86. 80 —
8. 72 4. 38 9. 96 & lt- 20. 00%
bR / sRb b -0. 002 / 0. 021 0. 010 / 0. 012 0. 031 / 0. 021 bR& lt- 1,812 sbR b
aR / sR 90. 57 / 2. 20 95. 97 / 1. 21 84. 06 / 2. 19 aR& lt- 1,812 s R a
I100-RI 9. 60 3. 20 13. 20 & lt- 6. 40%
linearity
ak -0. 023 -1. 193 1. 721 -1. 114 -2,050 -0,914 -3,321 -0,951 -0,021 a& lt- 2,015 s a a& lt- 8,53%
sk a 1. 476 2. 558 2. 448 2. 195 3,626 2,032 2,454 2,637 2,712
bk 1. 027 1. 032 0. 990 1. 037 1,045 1,026 1,063 1,041 1,019
skb 0. 013 0. 023 0. 022 0. 020 0,032 0,018 0,022 0,024 0,024
RSDk0 1. 747 3. 026 2. 897 2. 598 4,290 2,404 2,903 3,121 3,209 & lt- 9,93%
Rk 0. 9996 0. 9988 0. 9988 0. 9991 0,9976 0,9992 0,9989 0,9987 0,9986 & gt- 0,9830
accuracy
Zk 102. 51 102. 34 102. 36 102. 78 102,61 101,80 101,68 103,12 102,75 —
5k 2. 51 2. 34 2. 36 2. 78 2,61 1,80 1,68 3,12 2,75 & lt- 6,40%
Z'-ntra 102. 40 102. 40 102. 52 —
finira 2. 40 2. 40 2. 52 & lt- 6. 40%
precision
RSDkz z 2. 95 3. 44 6. 44 2. 93 3,72 2,75 4,68 3,37 4,41 —
A z 5. 73 6. 68 12. 51 5. 69 7,23 5,34 9,09 6,55 8,57 & lt- 20,00%
RSDintra z 4. 55 3. 16 4. 19 —
A^ntra z 7. 85 5. 45 7. 23 & lt- 20. 00%
? о
о ?
тз с & lt-0
a
о ф
й-
(О ф
. С

analysis of the model samples- g = 7. k = 3 (3 runs — 3 days) I
crp, e = ArplB = 25,50,75,100,125,150,175%

analysis of the blank-samples 1- n = 5 -> Ablgnk
RSD (blank} = -^r¦ 100%. %2
4J f (95%, n-1)
'-A™ = 0.1 (Am / 0−32 & lt- 0. 1), = 10. 32 (At"* 10−32 & gt- 0.1 J if unsatisfied-n is increased
) * sample & quot-25H
, 10o% & lt- max6J5% = 8. 00%3- 5,
25% '-
^(unsatisfied — tfra nent stage
¦100% & lt- maxo™ = 6. 40%3
analysis of the blank-samples 2- n = 3 -& gt- Aprocedm
mcedim 4
^procedure!25*4) Z Tssmple & lt- 0. 32 * 6(^(25%)
4il unsatisfied — t is necessary to modify the sample preparation procedure, i (satisfied — the value of/W is considered when calculating
recovery determination (for 25%, 50%, 100%, 175% - n = 4)
R, =-V ¦ 100%- R --
1) Д",=((95%Л-п-
¦n-1 Щ

n-k
& lt- maxi., =20. 00%
2) R = a* + bR-X -" afi- s, R- ?& gt-B- s*- a& quot- & gt- f (95%-A n~2) s8R- b* & lt- i (95%-/t -n-2)-sRb
3) |l00-ff|& lt- max5 = 6. 4Q%5 sif satisfied — the value of R is considered when calculating

r normalization of the obtained data
Qsampie
Xifact = 7 Г '-100%- Cfefefence = Степке '- & quot-
reference a sample
'-%="--100%- A^
reference 4ilulion coefficient
A ¦ R
reference 100
f calculation of linear dependence parameters'-^ within-run
Yk= ak +bk ¦ X& quot- a*- sj- bk- sj- RSDj- R*
RSD0 & lt- 9. 93% Rc & gt- 0. 9830
. a: 1) & lt- i (95%-g-2)'-Sa- 2) & lt- 8. 53%.
С
accuracy and precision
J
e within-run & gt-
Zf=i. 100%
л им
Akz = f (95%, g -1) -RSDii & lt-maxA, s = 20. 00% v 5& quot- = ?100-Z'-kmaxS = 6. 40%
У Zk
^ intra _ / * I
kg
between-run ¦100%- RSDf =
_ l?(RSPk)2
Af = f (95% ¦ g -1) ¦ RSDf & lt- max = 20. 00%

max б =6. 40%
Fig. 4. The stages of validation of spectrophotometry methods of captopril determination in blood using model samples.
At the second stage the methods validation was carried isolation from blood — not less than 90% - by the results of
out using model samples (fig. 4). recovery study. The method with acetonitrile application is
The total results of validation are presented in table 2. characterized by the best extraction efficiency.
The results of specificity study show that carrying out cap- The values reproducibility for recovery and blank-samples
topril isolation from blood using amphiphylic solvents pro- absorbance satisfy the acceptability criteria for all variants
vides low contribution of biological matrix components into of the methods. The absorbance values obtained for the
the absorbance of the sample to be analysed. It is possible blank-samples 2 are the evidence of the correct choice of
to point to the conclusion about high efficiency of captopril sample preparation procedure for all considered cases.
On the whole, all examined methods are characterized by the acceptable parameters of linearity, accuracy and precision, and the obtained data are the evidence of application possibility of the method of standard for captopril spectrophotometric determination in blood by reaction with the Ellman reagent.
For the method with acetonitrile application processing the results of experiment was carried out both with correction by the R value and without it. It was necessary to note that absence of such correction did not lead to worsening of the method validation parameters, but the method systematic error changed its sign (Zintel= 99. 09%).
References
1. Tiwari, G. (2010) Bioanalytical method validation: an updated review. Pharm. Methods, 1(1), 25−38. doi: 10. 4103/22 294 708. 72 226.
2. Moffat, A. C., Osselton, M. D., & amp- Widdop B. (Eds.) (2011) Clarke'-s analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem material. London: Pharmaceutical Press.
3. (2009) 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 on Drugs and Crime, Laboratory and Scientific Section. New York, United Nations.
4. (2013) Standard Practices for Method Validation in Forensic Toxicology. Scientific Working Group for Forensic Toxicology (SWGTOX).
5. Bolotov, V. V., Shovkovaya, Z. V., Merzlikin, S. I., Klimenko, L. Yu. (2007) Spektrofotometrychne vyznachennia kaptoprylu za dopomohoiu fotometrychnoi reaktsii z reaktyvom Ellmana [Spectrophotometric determination of captopril by the help of photometric reaction with the Ellman reagent]. Zhurnal orhan-ichnoi ta farmatsevtychnoikhimii, 4(20), 63−66. [in Ukrainian].
6. Klimenko, L. Yu. (2014) Razrabotka podkhodov k opre-deleniyu linejnosti, pravil'-nosti i precizionnosti UF-spektro-fotometricheskikh metodik kolichestvennogo opredeleniya metodom standarta v sudebno-toksikologicheskom analize [Development of approaches to determination of linearity,
Conclusions
Thus, we have developed the series of spectrophotometric methods of captopril quantitative determination in blood by reaction with the Ellman reagent using amphiphylic solvents (isopropanol, acetonitrile, methanol) for analyte isolation from matrix under the conditions of aqueous phase saturation by ammonium sulphate. Acetonitrile application in the acid medium (pH = 2) is optimal — contribution of matrix components into the absorbance of the sample to be analysed does not exceed 10%, extraction efficiency is ~97%.
Validation of the developed methods has been carried out using the approaches offered before by us and the possibility of the method of standard application for determination has been shown.
accuracy and precision of UV-spectrophotometric methods of quantitative determination by the method of standard in forensic and toxicological analysis]. Farmaciya Kazakhstana, 4, 31−35. [in Russian].
7. Klimenko, L. Yu., Petyunin, G. P., Kostina, T. A. (2013) Pod-khody k opredeleniyu specifichnosti/selektivnosti pri validacii UF-spektrofotometricheskikh metodik kolichestvennogo opre-deleniya v sudebno-toksikologicheskom analize [Approaches to determination of specificity/selectivity when validating UV-spectrophotometric methods of quantitative determination in forensic and toxicological analysis]. Farmaciya Kazakhstana, 8, 53−56. [in Russian].
8. Klimenk, L. Yu., Trut, S. M., Petyunin, G. P., & amp- Ivanchuk, I. M. (2013)Validation of UV-spectrophotometric methods of quantitative determination in forensic and toxicological analysis: recovery. Farmaciya Kazakhstana, 12, 42−48. [in Kazakhstan].
9. Shovkova, Z. V, Bolotov, V. V., Merzlikin, S. I., & amp- Klimenko, L. Yu. (2010) Rozrobka metodyk izoliuvannia kaptoprylu iz biolohichnykh ridyn orhanizmu [Development of captoprile isolation methods from organism biological liquids]. Zaporozhskij medicinskijzhurnal, 6, 84−87. [in Ukrainian].
10. Gerasimov, D. A. (2014) Khimiko-toksikologicheskoe issledo-vanie nimesulida i blizkikh po structure soedinenij (Dis.. kand. farm. nauk). [Chemical and toxicological research ofnimesulide and similar compounds by structure]. Cand. farm. sci. diss.]. Kursk. [in Russian].
Information about authors:
Klimenko Lina Yu., candidate of pharmaceutical sciences, assistant professor of analytical chemistry department, National University of Pharmacy, E-mail: lynnne2@ukr. net.
Showkova Zoya V., candidate of pharmaceutical sciences, assistant professor, assistant professor of toxicological chemistry department- National University of Pharmacy.
Mykytenko Olena Ye., candidate of pharmaceutical sciences, assistant professor of analytical chemistry department, National University of Pharmacy.
Kaplaushenko Andrew G., doctor of pharmacy, assistant professor, head of physical and colloidal department, Zaporizhzhya State Medical University.
Hagmmga b pega^iro 29. 09. 2014 p.

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