Comparative Study of Captopril Derivatization Reaction by LC-UV , LC-MS and CE-UV Methods

The LC-UV, LC-MS and CE-UV study of chemical reaction between captopril and p-bromophenacyl bromide as derivatizing reagent is reported. During transformation of captopril, its thiol group is involved and the reaction is irreversible. Neutral or alkaline environments favor derivatization. The yield of reaction increases linear with the concentration of the reagent, while changes in temperature do not influence it significantly. Kinetic studies show that derivatization of captopril flows quickly, reaching a plateau after 30 minutes. Based on the study of these effects some important parameters such as derivatizing agent concentration, reaction time, temperature and pH were confined. The results were used for development of captopril determination methods both in pharmaceutical formulations and biological samples. (doi: 10.5562/cca1787)


INTRODUCTION
Captopril (CAP) is an angiotensin converting enzyme inhibitor, used as antihypertensive agent.It is common that ACE inhibitors have low UV absorbtivity values and besides that captopril is very instable, undergoes to oxidation, the degradation product being the dimer. 1 Moreover, captopril also binds to endogenous compounds (cysteine, glutathione).3][4] To improve detection and to stabilize the compound a derivatizing agent can be used.6][7][8] The resulted product can be determined at a more specific wavelength (λ = 260 nm).The possible reaction between the derivatizing agent and captopril involves the -SH group of the latter and the carbocation of the alkylhalide, 9 resulting a derivative with greater molecular mass and less polarity (Figure 1).
The aim of our study was to optimize several parameters of the derivatization reaction to get a good yield of transformation, to use these parameters in sample processing and to apply the derivatization in deter-mination methods of captopril from biological matrices or pharmaceutical preparations.

Materials and Methods
All the reagents were of analytical or reagent grade purity.The derivatization reaction was followed by CE-UV, LC-UV and LC-MS methods.For preliminary studies a low cost CE-UV method was developed: The instrument was an Agilent 6100 CE System, with an uncoated capillary (l = 38 cm, 50 µm i.d.).The temperature was set at 23 and 40 °C, buffer solution used KH 2 PO 4 (c = 20 mmol dm -3 ) + NaOH (c = 0.1 mol dm -3 ), pH = 7.7.Polarity: positive, voltage: 20 kV, Injection: pressure of 5 mbar applied during 5 s.UV detection was performed at 205 and 260 nm.
For HPLC-UV studies the following assembly was used.The instrument was an Agilent 1100 series, chromatographic column was a Zorbax SB-C8 Solvent Saver Plus, l = 3 × 100 mm, 3.5 m i.d., the column temperature was set at 35 C.Mobile phase: phosphoric acid (c = 15 mmol dm -3 ) in water-acetonitrile (w = 60-40 %), flow: 1 mL min -1 , with gradient elution as described in Table 1.
The injection volume was 10 µL, the detector was set at 205 and 260 nm.
For the LC-MS method, the liquid chromatograph was an Agilent 1200 series, equipped with a Chromolith Performance 100 × 4.6 mm, 3 µm i.d.column (Merck).The column temperature was set at 30 C.The mobile phase consisted in a mixture between A: formic acid 0.1 % in water; B: acetonitrile in isocratic elution A : B = 60 : 40 %, flow 1 mL min -1 .The injection volume was 20 µL.The mass spectrometer, IonTrap SL 1100 series (Agilent), used an ionization source ESI+.The detection was in MRM mode, the monitored ions were 216, 253, 255, 368, 370 m/z fragmented from 415 m/z parent ion of the derivative.
Extraction of captopril from tablets: 20 tablets, each containing 25 mg captopril, were weighed and the average mass of one tablet was determined.A quantity of powder, corresponding to a content of 10 mg captopril, was extracted with approximatelly 7 ml water in a 10 mL volumetric flask in the ultrasonic bath (for 10 minutes), then brought to volume with water and filtered through the filter with the pore size of 0.45 µm.The resulted solution (γ = 1 mg mL -1 ) was diluted 20 fold with water to reach the desired captopril concentration of 50 µg mL -1 (0.5 mL of initial captopril solution was dilluted with water in 10 mL volumetric flask).The derivatization reaction was performed as follows: 200 µL from this sample was mixed with 20 µL 0.1 M KOH and 40 µL RD.After 15 minutes the mixture was neutralized with 30 µL of 1 M HCl, then injected to HPLC.
Sample preparation from plasma samples: Standard and test plasma samples were prepared as follows in order to be chromatographically analyzed.In an Eppendorf tube 0.2 mL plasma with 40 L RD solution was vortex-mixed for 30 seconds.After 30 minutes, while the derivatization reaction was completed, the tube was shaken again for 30 seconds with 0.3 mL methanol, and then centrifuged for 10 minutes at 14000 rpm.The supernatant was transferred in an autosampler vial and 20 L were injected into the LC-MS system.

RESULTS AND DISCUSSION
The electrophoretic method was used only for preliminary studies, to observe whether the reaction occurs or not.Firstly the electropherograms of the two reactants (CAP and p-BPB) were recorded, then those of their mixture, at 30 minutes after mixing.This electropherogram shows three peaks: the p-BPB being neutral migrates together with EOF, captopril, being the most negatively charged, is the last that migrates.The third peak that appeared is the derivative's one, which migrates in front of captopril, being less negatively charged and more voluminous (Figure 2).The presence of three peaks on the electropherogram shows that the reaction occurs and the product (derivative) is formed.
For further studies we switched to an HPLC-UV instrument, which gives better reproducibility for quantitative data and better determination of lower concentrations.With this method the parameters of derivatization reaction were optimized.

The Captopril: Derivatizing Agent Ratio
The optimum molar ratio was found to be CAP : p-BPB = 1 : 1.4.

The pH of the Reaction Medium
The reaction is favored by alkaline medium but it also occurs with acceptable rate at neutral pH, which is important for plasma sample analysis (Figures 3 and 4).

Reacting Time
The time necessary for a complete transformation is about 30 minutes at neutral pH, in alkaline medium the reaction occurs instantaneously (3-5 minutes).
The reaction order can be determined by the graphical method, plotting the peak area y / mAU min (which is proportional to the concentration of analyte) vs. time.The best fit to our data was achieved using a three parameter logarithmic function (Figure 5 Considering peak area max / 2 = 2500 / 2 = 1250 mAU min, using the obtained equation, the half-life time can be calculated, being t 1/2 = 2.8 min.This value is in good agreement with the experimental data (in approximately 3 minutes the half of the initial quantity of captopril is derivatized).
The reaction rate can be expressed as the integrated form of the above equation, in our case:     By HPLC-UV method a good separation of the derivative was achieved even in the presence of plasma matrix constituents.The detection of the derivative at 260 nm is much more selective than of captopril at 205 nm (Figure 6).
The detection limits calculated were minimum 1.0 g mL -1 for underivatized Captopril at 205 nm, and 0.1 g mL -1 in case of derivative, at 260 nm.So the specificity of wavelength allows 10 times lower detection limit for captopril as derivative (Figure 7).
The developed HPLC-UV method is suitable for determination of captopril from pharmaceutical formulations, e.g.tablets.Linearity of the method was verified in the 12-100 g mL -1 concentration range, with six calibration standards, the results are presented in Table 2.
The method has good accuracy and precision in the linearity range, Er and CV found to be below 3 % (Table 3).
The captopril content from tablets (C CAT , expressed in mg per tablet) is calculated using the sample concentration determined from the calibration curve (γ cc, expressed in g mL -1 ), the average mass (m mean /g) of the 20 tablets, the amount of sample taken (m sample /g), and taking into account also the dilution that was made: where γ cc = 48.84g mL -1 ; m mean = 0.14 g; m sample = 0.056 g (Figure 8).Substituting known values into the formula, the result is C CAT = 24.42mg, which corresponds to official regulations, assay of content in tablets ± 7.5 %, which means that captopril content has to be in the range of 23.125 -26.875 mg.
Taking in consideration the detection limits from HPLC-UV method, we can conclude that this method is still not suitable for bioequivalence and pharmacokinetic studies, where the plasma concentration of captopril is only of ng mL -1 (pg mL -1 injected to column) magnitude.This low-concentration problem can be solved by two ways: (i) Purification and concentration of samples by solid phase extraction (SPE) or liquid-liquid extraction (LL), but both are time consuming and very laborous.(ii) Use of more sensitive, specific and efficient detection mode, like MS or MS/MS.
The second way was considered being more efficient, so for further experiments an LC-MS method was used.
The MS/MS spectrum of the derivative is presented in Figure 9.The monitored ions were the MRM 216, 253, 255, 368 and 370 m/z, fragmented from the parent ion 415 m/z.
This HPLC-MS/MS method was able to quantify free captopril in human plasma after oral administration of a single dose of 100 mg captopril by applying a simple protein precipitation.Despite the sensitivity of the LC-MS/MS method, prior derivatization is needed in order to prevent captopril disulphide formation during sample processing and storage.The developed and validated method was used for bioequivalence investigation of two oral medicinal products containing captopril on a large number of human plasma samples.The results of the study were published previously by us. 10

CONCLUSIONS
An LC-UV, LC-MS and CE-UV study of chemical reaction between captopril and p-bromophenacyl bromide as derivatizing reagent was reported.During transformation of captopril, its thiol group is involved and the reaction is irreversible.The reaction occurs at room temperature in alkaline and neutral media as well, reaching a plateau after 30 minutes at neutral pH.The reaction follows a complex kinetic; the calculated half-life time (2.8 minutes) corresponds to the experimentally observed value.The optimized method of captopril determination was successfully applied to pharmaceutical formulations (HPLC-UV-25 mg tablets), and biological samples (bioequivalence study, HPLC-MS human plasma).

Figure 1 .
Figure 1.The coupling reaction between CAP and p-BPB, resulting a M r = 414 product.
and the rate constant: k = b = 539 AU.

Figure 4 .
Figure 4. Kinetic profile of the reaction at neutral pH = 7.4 (achieved with ammonium formate or KH 2 PO 4 + NaOH buffer).

Table 1 .
Gradient elution during HPLC-UV run

Table 2 .
Calibration curves in analytical runs (peak area = a × c + b; n = 4 series)