This paper demonstrates colorimetric visible spectrophotometric quantification methods for amino acid, namely, tranexamic acid and pregabalin. Both drugs contain the amino group, and when they are reacted with 2,4-dinitrophenol and 2,4,6-trinitrophenol, they give rise to yellow colored complexes showing absorption maximum at 418 nm and 425 nm, respectively, based on the Lewis acid base reaction. Detailed optimization process and stoichiometric studies were conducted along with investigation of thermodynamic features, that is, association constant and standard free energy changes. The method was linear over the concentration range of 0.02–200
Pregabalin (PG) chemically is 3-amino methyl hexanoic acid, with the chemical formula C8H17NO2. Its structural and pharmacological features correspond to the mammalian neurotransmitter gamma-aminobutyric acid (GABA), and it is primarily used as anticonvulsant drug. However, its effects are much broader being analgesic, antiepileptic, antidiabetic, and anti-inflammatory drug. Further more, it has been recommended for gastrointestinal damage, alcoholism, and insomnia [
Structure of PG and TXA.
PG is not yet part of any pharmacopoeial monograph. However, analytical method reported for PG includes spectrophotometry [
A glance over the literature revealed few reports regarding charge transfer complexes for PG and TXA. A structural analogous of PG and TXA gabapentin has been determined by colorimetry-visible spectrophotometry by reacting with iodine, chloranil, chloranilic acid, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, tetracyanoethylene, methyl orange, hydroxy benzaldehyde, picric acid and with ninhydrin [
There is no report on TXA and PG determination which utilizes TNP or DNP reagents. Most of the reported spectrometric methods are insensitive or need complicated extractions and heating/cooling procedures or use reagents which do not produce linear response. Moreover, most of the methods are based on the absorbance in near UV region and thus specificity is questioned. The method under proposal is highly selective and extremely simple, so it can be usefully adopted for routine analysis in quality control laboratories. The proposed methods are accompanied by the reaction of amine group of amino acid with hydroxyl group, activated by neighboring nitro groups of both TNP and DNP. As a result, yellow color complexes are formed on simple addition of the two reagents and no extra process is required. Thermodynamics features in respect of association constant and standard free energy changes have been evaluated for both reactions. Both of methods have been proved to be very useful from routine quality control prospective and can be considered as superior to most of the published methods with respect to pace, ease, low cost, and sensitivity.
A UV-Visible Shimadzu Spectrophotometer 1601 with 1 cm path length quartz cells controlled by Shimadzu UV Probe 3.9 version software was used. MS Excel sheet was used to evaluate
Analytical grade reagents were used. Pregabalin and tranexamic acid pure drugs were a kind gift from a local pharmaceutical agency having 99% plus purity. Tranex capsules 250 and 500 mg and Syngab capsules 200 mg, 50 mg, and 100 mg (Atco Laboratories Ltd., Karachi, Pakistan) were procured from the market. TNP and DNP were acquired from Merck Sigma Aldrich, Germany. Microcrystalline cellulose (Avicel pH 101, maize starch, magnesium stearate, PVP, Aerosil R-200, crospovidone-X, HPMC (606), and PEG (6000)), titanium dioxide, and isopropyl alcohol were a gift from a local pharmaceutical agency. The entire chemicals were used according to their safety precautionary measures.
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Homogenous samples were obtained for each drug molecule. Bulk homogenous powder equivalent to 10 mg of pregabalin or tranexamic acid was dissolved in methanol by sonication for 15 min and shaking thoroughly for about 30–40 min. The samples were cooled down and diluted up to the mark level with methanol, mixed well, and filtered using a Whatman number 42 filter paper to give 100
10 mg of each PG and 10 mg of TXA were spiked individually with common excipients like magnesium stearate, HPMC (hydroxypropyl methylcellulose), glucose, pyrrolidone, lactose, talc powder, and starch. It was further processed as described under general procedures. Samples were subjected to multiple analyses and percent recovery was calculated.
This reaction is based on the proton transfer reaction from Lewis acid such as TNP and DNP to Lewis base such as TXA and PG, which produces intensely yellow color ion-pair complex as depicted by Saito and Matsunaga [
Mechanism of reaction.
Standard free energy changes
Utilizing the above equation,
ICH guideline was followed for the validation purpose and various experiments were performed [
Linearity is the ability of method to show absorbance correspondingly to the analytes concentration. Least square procedure was adopted to develop the regression equations which showed linear relation of the concentration of complex with absorbance, complying Lambert Beer’s law. Under these experimental set-ups, absorbance at the given wavelength was found to vary directly with the concentrations of both the donner and the acceptor molecules.
Accuracy of the analytical method is that the parameter confirms that the test results obtained with the method are close to the true or accepted values, while precision is the reproducibility of test result when the same homogenous sample is subjected to multiple testing. Solutions containing three different concentrations of pregabalin and tranexamic acid were arranged and investigated in triplicate for accuracy and precision.
Specificity is the quantification of the analytes in presence of component mixtures, excipients, and additives. The effect of common excipients and additives was evaluated by designing spiking experiments. Both TXA and PG were evaluated with common excipients at different concentration levels individually.
Both reactions were evaluated for LOD and LOQ values. The empirical formulas
The reaction of coloring reagent TNP or DNP as Lewis acids with amino acid (PG and TXA) as Lewis base afforded intense yellow charge transfer complex. The intensity in color is attributed to the formation of phenolate ion [
In order to achieve optimum experimental condition, various factors were evaluated including time, temperature, solvent of choice, and concentration of drugs and reagent. Those experimental factors which were affecting the absorbing abilities of the resulting complex were optimized to enhance selectivity and sensitivity. The reaction and stability of the complex were evaluated with respect to time in intervals at room temperature. Maximum absorbance was obtained in 5- to 10-minute time period and prolonged period of time did not affect the absorbance of the complex. However, a negative impact was noted when reaction mixture was heated especially beyond 60°C. Similarly, effect of coloring reagent was evaluated by adding it to the fixed concentration of drug substance. To 100
Spectrum of complexes and blanks.
Job’s continuous variations method suggested a 1 : 1 molar ratio for both reagents with both drugs. However, experimental design considered the drug molecules to be limiting reactants, so the chromogenic reagent was taken in slight excess in subsequent analysis in order to make the reaction drug concentration dependent and to counter any possible interference.
Complexes were studied in detail by applying Bensi-hilbrand theory. Association constant
Thermodynamic study.
Drug molecule | DNP | TNP | ||
---|---|---|---|---|
PG | TXA | PG | TXA | |
Molar absorptivity (Ma) | 1326.85 | 1337.35 | 1404.77 | 1418.36 |
|
1.51 × 103 | 1.62 × 103 | 2.74 × 103 | 2.86 × 103 |
|
−2.336 | −2.358 | −2.498 | −2.508 |
After development method validation studies were conducted in line with the above-mentioned protocol. Using linear regression equation, methods showed linear response in concentration range from 0.02 to 200
Linearity and range.
Parameter | DNP | TNP | ||
---|---|---|---|---|
PG | TXA | PG | TXA | |
Linearity range ( |
0.02–200 | 0.02–200 | 0.02–200 | 0.02–200 |
Correlation coefficient ( |
0.99962 | 0.99971 | 0.99948 | 0.99987 |
Slope ( |
0.01002 | 0.02035 | 0.01423 | 0.01553 |
Intercept ( |
−0.0012 | −0.0028 | 0.0021 | −0.0011 |
(LOD) ( |
0.0059 | 0.0094 | 0.0041 | 0.0075 |
(LOQ) ( |
0.0195 | 0.0302 | 0.0137 | 0.0269 |
Accuracy and precision.
Pregabalin (PG) | Tranexamic acid (TXA) | |||||
---|---|---|---|---|---|---|
50% nominal | 100% nominal | 150% nominal | 50% nominal | 100% nominal | 150% nominal | |
con. %recovery | con. %recovery | con. %recovery | con. %recovery | con. %recovery | con. %recovery | |
%l.c found %RSD | %l.c found %RSD | |||||
DNP | 98.10 1.235 | 101.68 0.525 | 101.93 0.235 | 99.84 0.545 | 99.84 0.412 | 101.85 0.864 |
TNP | 100.60 0.320 | 99.89 0.536 | 99.17 0.938 | 101.60 0.940 | 100.69 0.526 | 101.57 0.638 |
Recovery studies in commercial products.
Brand name (active molecule): Label claim | Label claim (%) ± S.D. | |
---|---|---|
DNP | TNP | |
Tranex (TXA): 250 mg | 101.23% ± 0.51 | 100.15% ± 0.61 |
Tranex (TXA): 500 mg | 99.95% ± 0.63 | 99.19% ± 0.92 |
|
||
Syngab (PG): 100 mg | 99.99% ± 0.77 | 99.91% ± 0.65 |
Syngab (PG): 200 mg | 101.47% ± 1.13 | 100.27% ± 1.22 |
General procedures described above were followed to determine content of capsule dosage pharmulations for both of the drugs. The projected colorimetric methods based on charge-transfer complexes were applied to determine PG and TXA, along with the reference methods 9 and 15 for PG and TXA, respectively, to evaluate the test results.
Taken (mg) | TNP | DNP | Reference method | |||
---|---|---|---|---|---|---|
PG found (mg) | TXA found (mg) | PG found (mg) | TXA found (mg) | PG [ |
TXA [ | |
100.1 | 100.2 | 100.6 | 101.6 | 100.7 | 100.3 | 101.1 |
101.1 | 99.8 | 100.3 | 98.6 | 99.4 | 100.8 | 99.8 |
100.2 | 100.1 | 99.8 | 100.9 | 100.3 | 99.7 | 101.2 |
100.4 | 100.2 | 99.6 | 99.1 | 98.7 | 99.3 | 100.7 |
100.3 | 100.6 | 100.6 | 98.4 | 97.9 | 99.5 | 100.4 |
99.6 | 100.5 | 101.4 | 97.5 | 98.1 | 100.5 | 100.3 |
MEAN | 100.23 | 100.38 | 99.35 | 99.13 | 100.01 | 100.58 |
SD | 0.288 | 0.646 | 1.576 | 1.184 | 0.601 | 0.527 |
RSD | 0.287 | 0.644 | 1.586 | 1.195 | 0.601 | 0.524 |
|
0.44 | 0.569 | 0.355 | 0.022 | ||
|
0.131 | 0.665 | 0.054 | 0.110 |
To estimate the quantity of tranexamic acid and pregabalin in commercial products, two simple and sensitive colorimetric methods were developed and validated. Picric acid and 2,4-dinitrophenol, the two coloring reagents, have been utilized to develop two simple, much more common but sensitive and selective visible range spectrophotometric methods for routine analysis of tranexamic and pregabalin in bulk raw material and finished or semifinished dosage form. The suggested methods are superior to the already established spectrophotometric methods in terms of simplicity.
The authors declare that they have no conflict of interests regarding the publication of this paper.