Pharmaceutical Chemistry Review Volume 1 2015 http://www.bacpl.org/J/pcr
Development and Validation of a Non‐ Aqueous Titrimetric Assay for the Determination of Pregabalin in Bulk Drug Luciana Petrone, Mariela Baldut, Silvina L. Bonafede, Laura D. Simionato, Adriana I. Segall Cátedra de Calidad de Medicamentos, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Junín 956, 1113 Buenos Aires, Argentina. Abstract New, accurate, sensitive and economical non‐aqueous volumetric method for the determination of pregabalin in raw material has been developed. The titrimetric method involves the reaction of perchloric acid with pregabalin and crystal violet was used as indicator to detect the end of the titration. The method was validated by following the analytical performance parameters suggested by the International Conference on Harmonization (ICH). The calibration curve was linear from 80 to 120 % with r = 0.9994. Statistical treatment of the experimental results indicated that the method is precise and accurate. The proposed method can be used for quality control assay of pregabalin in bulk drug. Keywords Pregabalin; Non‐Aqueous Titrimetry; Assay
Introduction Pregabalin (Fig 1) is (S)‐3‐(Aminomethyl)‐5‐methylhexanoic acid. It has been shown to be clinically effective and is approved for the treatment of postherpetic neuralgia, fibromyalgia, neuropathic pain associated with diabetic peripheral neuropathy, neuropathic pain associated with spinal cord injury, and as adjunctive therapy for adult patients with partial onset seizures in the United States. In Europe, pregabalin is approved for the treatment of central and peripheral neuropathic pain, generalized anxiety disorder, and also as adjunctive therapy for adult patients with partial onset seizures with or without secondary generalization. Pregabalin binds to the α2δ1 and α2δ2 subunits of voltage‐sensitive calcium channels with high affinity. Mutation of the α2δ1 subunit blocked pregabalin effects in neuro‐pathic pain and anxiety‐related assays; however, mutation of the α2δ2 subunit had no effect on anxiolytic activity [1]. The usual doses are capsules or tablets containing 25, 50, 75, 150 and 300 mg.
FIGURE 1 PREGABALIN
Pregabalin has been determined by spectrophotometric methods introducing a chromophoric group, in pure drug [2‐5] and with other drugs [6‐7], electrophoresis capilar [8‐9], HPTLC [10] and gas chromatography [11]. A literature survey revealed some high‐performance liquid chromatographic methods for the quantitation of pregabalin in biological fluids [12‐16] and in pharmaceutical products [17‐22] and using UPLC [23‐24]. Due to the need for an absolute analytical technique for pregabalin, we have developed this volumetric technique. The non‐aqueous titrations have become of considerable importance in pharmaceutical analysis because it is very simple and easily adoptable to determine active pharmaceutical ingredients, especially in the developing countries where modern and expensive instruments are not available.
Author to whom correspondence should be addressed: e-mail: asegall@ffyb.uba.ar.
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Pregabalin has two pKa values, 4.2 and 10.6, corresponding to the carboxylic acid and the amine groups, respectively. The method is based on the neutralization reaction of the primary amino group of pregabalin with acetous perchloric acid as titrant in anhydrous acetic acid medium. The end was detected visually using crystal violet as indicator. The titration reaction follows a 1:1 stoichiometry (Fig.2).
FIGURE 2 REACTION SCHEME
The present manuscript describes a simple, rapid, precise, accurate and economic volumetric method for the quantitation of pregabalin in raw material. The method was validated by following the analytical performance parameters suggested by the International Conference on Harmonization (ICH) [25]. Materials and Methods Apparatus A standard borosil burrets, pipetts, standard flasks, measuring cilindres and conical flasks are calibrated as per International Conference on Harmonization (ICH) guidelines [26]. Materials Pregabalin (99.6%) was obtained from Zhejiang Tiantai Aurisco Pharmaceutical Co. Ltd. (Zhejiang, China). Potassium biphthalate ACS grade, acidimetric standard, (Mallinckrodt Baker ink., France), Perchloric acid ACS grade (Merck Química Argentina, Argentina), Glacial acetic acid AR grade (Sintorgan, Argentina), Acetic anhydride (Mallinckrodt, USA), Crystal violet AR grade (BDH Standard Stain, UK.) 1) Perchloric Acid 0.1N in Glacial Acetic Acid Mix 8.5 mL of perchloric acid in 500 mL of glacial acetic acid and 21 mL of acetic anhydride, cool, add glacial acetic acid to make 1000 mL. Adjust the water content. 2) 1% Crystal Violet Dissolve 100 mg of crystal violet in 10 mL of glacial acetic acid. Standardization Place 700 mg accurately weight of potassium biphthalate previously dried at 120°C for 2 h at dissolve it in 50 mL of glacial acetic acid in a 250 mL conical flask. Add 2 drops of 1% crystal violet and titrate with perchloric acid solution until the violet color changes to emerald‐green color. Each 20.42 mg of potassium biphthalate is equivalent to 1 mL 0.1N of perchloric acid.
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Pharmaceutical Chemistry Review Volume 1 2015 http://www.bacpl.org/J/pcr
General Procedure 350 mg of pregabalin was accurately weighed, transferred to a 250 ml conical flask, and dissolved in 100 mL of glacial acetic acid. Stir it for 10 min. Add 2 drops of crystal violet TS and titrate with perchloric acid solution until the violet color changes to emerald‐green color. Each 15.92 mg of pregabalin is equivalent to 1 mL 0.1N of perchloric acid. Method Validation 1) Linearity The linearity of the volumetric method was determined by analysis of six replicates at 80%, 100% and 120% and three replicates at 90% and 110% of analyte concentration. 2) Precision Precision of the method was checked by carrying out six independent assays of pregabalin raw material. Intermediate precision was performed by analyzing the samples by two different analysts on different days. 3) Accuracy The accuracy was evaluated by the recovery studies at concentration levels of 80%, 100% and 120 % (6 samples each). The amount of pregabalin recovered in relation to the added amount was calculated. Results and Discussion The proposed procedure has been satisfactorily applied to the quantitation of pregabalin in raw material. The linearity of the volumetric method was determined by analysis of six replicates at 80%, 100% and 120% and three replicates at 90% and 110% of analyte concentration. The calibration curve showed good linearity over the concentration range. The correlation coefficient (“r”) value was 0.9994. Typically, the regression equation for the calibration curve was found to be y = 0.0563x + 0.0317. The linearity of the calibration graphs was validated by the high value of the correlation coefficient and the intercept value that was not statistically (p = 0.05) different from zero (Table 1 and Figure 3). TABLE 1 LINEARITY
% of nominal value 80 90 100 110 120
Weighed (mg) (RSD) 280.2 (0.2) 314.7 (0.0) 351.2 (0.5) 381.8 (0.2) 411.7 (0.1) Slope(a) 0.0563 ± 0.0009 Intercept(b) 0.0317 ± 0.4044
Vol cons (RSD) 15.8 (0.3) 17.8 (0.3) 19.8 (0.6) 21.8 (0.8) 23.2 (0.5)
Confidence limits of the slope (p= 0.05); Confidence limits of the intercept (p= 0.05)
a
b
FIGURE 3 LINEARITY
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www.bacpl.org/j/pcr Pharmaceutical Chemistry Review Volume 1 2015
The precision of an analytical procedure expresses the closeness of the agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The intra‐day precision of the volumetric method was performed by assaying the samples on two different days by two different analysts. The results were given both individually and as the average. For each precision assay the results were as follows: mean values 98.8 and 99.3 %, RSD 0.2% and 0.3%. “t” Test comparing two samples with 95% confidence for 10 degrees of freedom disclosed that both results were not significantly different inter se (tn‐2, :0.05) = 2.23 (Table 2). TABLE 2 INTERMEDIATE PRECISION
Analyst 1 Sample N°
Weighed (mg)
Percentage
Analyst 2 Sample N°
Weighed (mg)
Percentage
1
350.9
99.1
1
351.2
99.1
2
352.6
98.7
2
351.2
99.6
3
352.6
98.7
3
349.2
99.1
4
354.0
98.8
4
354.2
99.2
5
350.5
98.7
5
353.5
99.4
6
350.8
98.7
6
352.4
99.7
Mean RSD
351.9 0.4
98.8 0.2
352.0 0.5
99.3 0.3
The recovery range values were within the range 99.1‐100.3%. Method accuracy of the volumetric method was also demonstrated by plotting the amount of pregabalin measured against the amount present in the samples, both expressed in mg. Linear regression analysis rendered slopes not significantly different from 1 (t test p=0.05), intercepts not significantly different from zero (t test p=0.05) and r = 0.9997, the RSD was 0.3 (Table 3 and Figure 4). TABLE 3 ACCURACY
% of nominal value
Added amount (mg)
Found amount (mg)
280.5 80
280.2 279.6 280.7 280.6 279.8
279.0 279.0 279.0 279.0 279.0 280.8
351.2 100
351.2 349.2 354.2 353.5 352.4
120
Mean (n=18)
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347.9 349.6 346.1 351.4 351.4 351.4
Recovery (%)
RSD (%)
99.7
0.4
99.3
0.3
99.5
0.4
99.5
0.3
99.5 99.6 99.8 99.4 99.4 100.3 99.1 99.6 99.1 99.2 99.4 99.7
Average recovery (n=3)
411.0
407.9
99.2
411.8
409.7
99.5
412.2
413.2
100.2
411.7
409.7
99.5
412.0
409.7
99.4
411.2
407.9
99.2
Pharmaceutical Chemistry Review Volume 1 2015 http://www.bacpl.org/J/pcr
FIGURE 4 ACCURACY
Conclusions The developed non‐aqueous titrimetric method for the determination of pregabaline was sensitive, accurate and precise and hence can be used for the routine analysis of the active pharmaceutical ingredient in raw material. The volumetric method proposed is simple, rapid and inexpensive. ACKNOWLEDGEMENTS
This work was supported by grant 20020130100342BA to A. I. Segall from UBA and PIP Nº: 11420110100380 from CONICET. REFERENCES
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