CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.10 No.6, pp 274-280, 2017
1, 213
Abstract : UV-visible spectrophotometry analytical method was validated for detection and quantitative analysis of nefopam hydrochloride(NPH) in microspheres synthesized using poly-3-hydroxybutyrate and poly-ȭ-caprolactone. UV absorption maximum (λmax) of NPH in phosphate buffer (pH 7.4) was found 266 nm. Limit of detection (LOD) and limit of
ɑɕɁɎɔɉɆɉɃɁɔɉɏɎ (Ȭȯȱ) ɏɆ ȮȰȨ ɗɅɒɅ ɆɏɕɎɄ 16.75 ɁɎɄ 50.77 ȝɇ/ɍɌ, ɒɅɓɐɅɃɔɉɖɅɌə. ȲɅɐɅɁɔɁɂɉɌɉɔə,
intermediate precision and specificity of developed analytical method wereestablished(RSD< 2%). The analytical method was found robust at different wavelengths (± 6) and temperatures (± 20 oC) (RSD < 2%). It was concluded that developed and validated UV spectrophotometry analytical method can be employed for routine quantitative analysis of NPH in microspheres synthesized using poly-3-hydroxybutyrate and poly-ȭ-caprolactone. Keywords: Nefopam Hydrochloride, Poly-3-Hydroxybutyrate and Poly-ȭ-Caprolactone, Limit of Detection, Limit of Quantification, Intermediate Precision.
Spectroscopy is domain of science dealing with the analysis of interaction between electromagnetic radiation and matter. UV-Visible spectrophotometry is most widely applied method in pharmaceutical analysis for analysis of specific compound in precise and specific manner. In qualitative analysis, it quantifies the amount of ultraviolet or visible radiation absorbed by material in solution to determine the quantity of material. Quantitative spectrophotometric analysis is based upon Beer -Lambert law. Beer-Lambert law states that when beam of parallel monochromatic light is passed through a transparent cell containing a solution of an absorbing substance, intensity of light may decreases. Mathematically, Beer-Lambert law is indicated as:
!" Eq. (1)
ȷɈɅɒɅ, ʲȡʳ ɉɓ ɁɂɓɏɒɂɁɎɃɅ, ʲɁʳ ɉɓ Ɂɂɓɏɒɐɔɉɖɉɔə ɏɒ ɅɘɔɉɎɃɔɉɏɎ ɃɏɅɆɆɉɃɉɅɎɔ, ʲɂʳ ɉɓ ɐɁɔɈ ɌɅɎɇɔɈ ɏɆ ɒɁɄɉɁɔɉɏɎ ɔɈɒɏɕɇɈ
[1-3]
ɓɁɍɐɌɅ (Ƀɍ), ʲɃʳ ɉɓ ɃɏɎɃɅɎɔɒɁɔɉɏɎ ɏɆ ɓɏɌɕɔɅ ɉɎ ɓɏɌɕɔɉɏɎ .
Analytical method validation is the process to demonstrate that analytical procedure employed for specific test is suitable for its intended use and support identity, strength, quality, purity and potency of drug substances and drug products. Nefopam hydrochloride (NPH) is a potent non-opioid centrally acting analgesic of benzoxazocine class [4] . In this investigation, efforts were made to develop a simple and economic UV-visible spectrophotometric approach for detection and quantitative analysis of NPH in microspheressynthesized using poly-3-hydroxybutyrate and poly-ȭ-caprolactone. Developed analytical method was validated for appropriate system parameters as per guidelines of international conference on harmonization (ICH) to establish sensitivity (limit of detection and limit of quantification), repeatability, intermediate precision, specificity and
[5, 6]
robustness .
Preparation of Stock and Working Standard Solution
100 mg of NPH was accurately weighed and dissolved in 50 ml of phosphate buffer pH 7.4 in a volumetric flask to produce 2000 µg/ml solution. 25 ml solution was withdrawn and diluted to 50 ml with phosphate buffer, pH 7.4 to give standard stock solution having concentration of 1000 µg/ml. From stock solution, 2.5 ml solution was transferred into 10 ml volumetric flask and volume was made up to mark with phosphate buffer, pH 7.4 to produce 250 µg/ml working standard solutions.
Determination of Absorption Maxima (λmax) of NPH
ȡ ɗɏɒɋɉɎɇ ɓɔɁɎɄɁɒɄ ɓɏɌɕɔɉɏɎɓ ɏɆ ȮȰȨ (250 ȝɇ/ɍɌ) was prepared using phosphate buffer, pH 7.4 and 8 ɍɌ ɏɆ ɓɏɌɕɔɉɏɎ ɗɁɓ ɄɉɌɕɔɅɄ ɔɏ 10 ɍɌ ɗɉɔɈ ɐɈɏɓɐɈɁɔɅ ɂɕɆɆɅɒ, ɐȨ 7.4 ɔɏ ɏɂɔɁɉɎ 200 ȝɇ/ɍɌ ɒɅɆɅɒɅɎɃɅ ɓɏɌɕɔɉɏɎ. ȡɎ UV spectroscopic scanning (200-400 nm)was carried out with reference solution to determine λmax for detection of NPH using phosphate buffer, pH 7.4 as blank.
Calibration Curve of NPH
From 1000 µg/ml standard stock solution, aliquots (i.e. 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 mland 4 ml) were transferred into 10 ml volumetric flask and volume was adjusted with phosphate buffer, pH 7.4 to
[7, 8]
make dilutions having 50-400 µg/ml concentration and analyzed for absorbance .
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The limit of detection (LOD) and limit of quantification (LOQ) of NPH was evaluated from the slope
(S) of calibration curve and standard deviation of y-intercept of the regression equation using following equations:
Ȯ
Eq. (2)
ଵȮ
Eq. (3)
The LOD is lowest amount of analyte which can be detected in sample, but not necessarily quantities as an exact value while LOQ is minimum quantity that can be quantified by the instrument[9] .
Repeatability (intra-day precision)
The different concentrations of nefopam hydrochloride i.e. 150, 200 ɁɎɄ 250 ȝɇ/ɍɌ ɗɅɒɅ ɁɎɁɌəɚɅɄ Ɂɔ
[10, 11]
three different times within a day. The % RSD should be less than 2% for acceptable repeatability .
Intermediate Precision
Intermediate precision articulates within-laboratories variations: different days (inter-day), different analysts and different equipment. The different concentrations of nefopam hydrochloride i.e. 150, 200 and 250
[10, 11]
ȝɇ/ɍɌ ɗɅɒɅ ɁɎɁɌəɚɅɄ (% ȲȳȤ Ɍɉɍɉɔ: < 2%) ɏn three different days (inter-day precision) . 200 ȝɇ/ɍɌ ȮȰȨ
solutions were analyzed by different equipments (Systronics AU-2701, Ahmedabad, India; Systronics 2202, ȡɈɍɅɄɁɂɁɄ, ȩɎɄɉɁ) ɁɎɄ ɁɎɁɌəɓɔɓ ɉɎ ɓɉɘ ɔɒɉɁɌɓ. ȡɂɓɏɒɂɁɎɃɅʳɓ ɗɅɒɅ ɓɕɂɊɅɃɔɅɄ ɔɏ ɃɁɌɃɕɌation for mean, standard deviation, % relative standard deviation (% RSD limit: < 2%), standard error and % coefficient of variation [5] .
Specificity
30 mg of NPH was mixed with 100% (30 mg), 200% (60 mg), 300% (90 mg), 400% (120 mg) and 500% (150 mg) of excipients (polyhydroxybutyrate and polycaprolactone) and analyzed for % recovery of NPH. The accepted limits of % recovery and RSD for validating specificity are 98% -102% and < 2%, respectively [11] .
Robustness
Robustness of UV spectrophotometry analytiɃɁɌ ɍɅɔɈɏɄ ɗɁɓ ɄɅɔɅɒɍɉɎɅɄ ɂə ɁɎɁɌəɚɉɎɇ ɔɈɅ 250 ȝɇ/ɍɌ NPH solutions at different temperatures i.e. 25±20oȣ ɁɎɄ ɗɁɖɅɌɅɎɇɔɈɓ (șmax) i.e. 260 ± 6 nm. % RSD acceptance limit is < 2% [5] . UV Spectrophotometry Method Validation Absorption Maxima (λmax) of NPH
Fig. 1 depicts UV scan of solution of NPH solution in phosphate buffer acquired using UV spectroscopy Ɂɔ șmax of 266 nm.
Fig. 1 UV scans of NPH solution in phosphate buffer, pH 7.4.
Calibration Curve of NPH
Calibration curve of NPH was acquired using UV spectrophotometric technique by plotting a graph between concentrations of NPH vs. absorbance value obtained at 266 nm (Fig. 2). Statistical analysis of calibration curve of NPH was performed by curve linear regression. Regression coefficient and P value was found 0.9976 and 0.008694 (P* < 0.05), respectively, which illustrated goodness of fit as well as statistical significance of proposed method (Table 1).
Fig. 2 Calibration curve of NPH in phosphate buffer, pH 7.4 using UV spectroscopy. Table 1 Linear regression statistical data of calibration curve for NPH.
Parameter | Value |
Best-fit values | |
Slope | 0.001334 ± 0.00002683 |
Y-intercept when X=0.0 | 0.1171 ± 0.006774 |
X-intercept when Y=0.0 | -87.79 |
1/slope | 749.9 |
95% Confidence intervals | |
Slope | 0.001268 to 0.001399 |
Y-intercept when X=0.0 | 0.1005 to 0.1336 |
X-intercept when Y=0.0 | -105.0 to -72.11 |
Goodness of fit | |
R square | 0.9976 |
P value | 0.008694 |
[9, 12]
Analytical Method Validation Parameters Limit of Detection (LOD) and Limit of Quantitation (LOQ)
LOD and LOQ of NPH in phosphate buffer, pH 7.4 ɗɅɒɅ ɆɏɕɎɄ 16.75 ɁɎɄ 50.77 ȝɇ/ɍɌ, ɒɅɓɐɅɃɔɉɖɅɌə
Repeatability
ȴɈɅ % ȲȳȤ Ɇɏɒ ɁɂɓɏɒɂɁɎɃɅ ɖɁɌɕɅɓ ɏɆ 150, 200 ɁɎɄ 250 ȝɇ/ɍɌ ȮȰȨ ɓɏɌɕɔɉɏɎɓ Ɂɔ ɔɈɒɅɅ ɄɉɆɆɅɒɅɎɔ ɔɉɍɅ
periods within a day was found to be 1.14, 0.64 and 0.67 % (< 2%), which validated repeatability of analytical
[11, 13, 14]
method (Table 2) .
Intermediate Precision
ȴɈɅ % ȲȳȤ Ɇɏɒ ɁɂɓɏɒɂɁɎɃɅ ɖɁɌɕɅɓ ɏɆ 150, 200 ɁɎɄ 250 ȝɇ/ɍɌ ȮȰȨ ɓɏɌɕɔɉɏɎɓ ɏɎ ɔɈɒɅɅ ɄɉɆɆɅɒɅɎɔ ɄɁəɓ
was found 0.79, 0.51 and 0.56 % (< 2%), which validated inter-day precision of analytical method (Table 2)[10,
11, 14]
.
Table 2 Repeatability and inter-day precision determined for three different concentrations of nefopam hydrochloride (n = 3).
Conc. (μg/ml) | Repeatability | Inter-day precision | ||
---|---|---|---|---|
Absorbance measured | % RSD | Absorbance measured | % RSD | |
150 | 0.315 | 1.14 | 0.3167 | 0.79 |
200 | 0.3937 | 0.64 | 0.395 | 0.51 |
250 | 0.446 | 0.67 | 0.4457 | 0.56 |
% RSD of absorbance values of sample solutions analyzed by analyst-1, analyst-2, equipment-1 and equipment-2 was found 0.46250%, 0.55647%, 0.33063% and 0.48463%, respectively. The % RSD values were < 2% which indicated intermediate precision of developed analytical method (Table 3) [5] .
Table 3 Intermediate precision data of UV spectrophotometry analytical method.
Condition | Trials | Absorbance | Mean | SD | % RSD | Std. error | % CV |
---|---|---|---|---|---|---|---|
Analyst-1 | 1 | 0.4430 | 0.4467 | 0.002066 | 0.46250 | 0.0008433 | 0.46 |
2 | 0.4480 | ||||||
3 | 0.4470 | ||||||
4 | 0.4490 | ||||||
5 | 0.4460 | ||||||
6 | 0.4470 | ||||||
Analyst-2 | 1 | 0.4480 | 0.4462 | 0.002483 | 0.55647 | 0.001014 | 0.56 |
2 | 0.4460 | ||||||
3 | 0.4420 | ||||||
4 | 0.4470 | ||||||
5 | 0.4450 | ||||||
6 | 0.4490 | ||||||
Equipment-1 | 1 | 0.4450 | 0.4452 | 0.001472 | 0.33063 | 0.0006009 | 0.33 |
2 | 0.4470 | ||||||
3 | 0.4460 | ||||||
4 | 0.4430 | ||||||
5 | 0.4460 | ||||||
6 | 0.4440 | ||||||
Equipment-2 | 1 | 0.4430 | 0.4457 | 0.002160 | 0.48463 | 0.0008819 | 0.48 |
2 | 0.4490 | ||||||
3 | 0.4440 | ||||||
4 | 0.4450 | ||||||
5 | 0.4460 | ||||||
6 | 0.4470 |
Specificity
Specificity of UV spectrophotometry analytical method was determined by analyzing NPH in presence and absence of excipients (poly-3-hydroxybutyrate and poly-ȭ-caprolactone). Mean recovery of NPH was found
99.93 % which was within accepted limit (98 % -102 %). The % RSD was found 0.8644 % (< 2 %) which
[11, 15]
validated specificity of analytical method (Table 4) .
Table 4 Specificity data of UV spectrophotometry analytical procedure.
PHB:PCL * (1:1) | NPH input (mg) | NPH recovered (mg) | NPH Recovered (%) | Mean Recovered | Statistical analysis |
---|---|---|---|---|---|
100 % | 30 | 30.2 | 100.67 | 99.93 % | Mean = 99.93 SD= 0.8638 % RSD = 0.8644 Std. error = 0.386 % CV = 0.86% |
200 % | 30 | 30.3 | 101.00 | ||
300 % | 30 | 29.8 | 99.33 | ||
400 % | 30 | 29.9 | 99.67 | ||
500 % | 30 | 29.7 | 99.00 |
*
PHB: Poly-3-hydroxybutyrate; PCL: Poly-ȭ-caprolactone
Robustness
% RSD of absorbance values of sample solutions analyzed at different wavelengths and temperatures was found 0.3424% and 0.5634%, respectively. The % RSD values were < 2% which indicated that proposed analytical method remained unaffected by small but deliberate variations in method parameters and provided an
[5, 14]
indication of its reliability during normal usage (Table 5) .Validation parameters of spectrophotometric method are shown in Table 6.
Table 5 Robustness studies of UV spectrophotometry analytical method.
Condition | Parameter | Absorbance | Mean | SD | % RSD | Std. error | % CV |
---|---|---|---|---|---|---|---|
Change in | 260 nm | 0.448 | 0.4463 | 0.00152 | 0.3424 | 0.0008819 | 0.34 |
Wavelength | 266 nm | 0.445 | |||||
272 nm | 0.446 | ||||||
Change in | 5 oC | 0.447 | 0.4467 | 0.00251 | 0.5634 | 0.001453 | 0.56 |
temperature | 25oC | 0.449 | |||||
45 oC | 0.444 |
Table 6 UV spectrophotometry analytical method validation parameters.
Parameter | Result |
șmax (nm) | 266 |
Regression equation (y= mx + c) | y = 0.001x + 0.117 |
Regression coefficient (r2) | 0.9976 |
Limit of detection (LOD) | 16.75 ȝɇ/ɍɌ |
Limit of quantitation (LOQ) | 50.77 ȝɇ /ɍɌ |
Repeatability indicated by % RSDfor NPH (150, 200 and 250 ȝɇ/ɍɌ) | 1.14, 0.64 and 0.67 % |
Intermediate precision | |
% ȲȳȤ Ɇɏɒ ȮȰȨ, 150, 200 ɁɎɄ 250 ȝɇ/ɍɌ (ȩɎɔɅɒ-day) | 0.79, 0.51 and 0.56 % |
Indicated by % RSD (analyst-I, analyst-2) | 0.46250, 0.55647 % |
Indicated by % RSD (equipment-1, equipment-2) | 0.330, 0.484 % |
Specificity indicated by % recovery | 99.93 % |
ȲɏɂɕɓɔɎɅɓɓ ɉɎɄɉɃɁɔɅɄ ɂə % ȲȳȤ (șmax, ± 6 nm) | 0.3424 % |
Robustness indicated by % RSD (Temp. ± 20oC) | 0.5634 % |
UV absorption maximum (λmax) of NPH in phosphate buffer (pH 7.4) was found 266 nm. LOD and Ȭȯȱ ɏɆ ȮȰȨ ɗɅɒɅ ɆɏɕɎɄ 16.75 ɁɎɄ 50.77 ȝɇ/ɍɌ, ɒɅɓɐɅɃɔɉɖɅɌə. The repeatability and intermediate precision of developed analytical method was validated through % RSD(< 2%). The % RSD was found 0.8644 % (< 2 %) on analyzing NPH in presence and absence of poly-3-hydroxybutyrate and poly-ȭ-caprolactone which validated specificity of analytical method. The % RSD values were found < 2% when sample solutions were analyzed at different wavelengths (± 6) and temperatures (± 20 oC) which validated the robustness of method.It was concluded that developed and validated UV spectrophotometry analytical method can be employed for routine analysis and quantitative evaluation of NPH in microspheres synthesized using poly-3-hydroxybutyrate and poly-ȭ-caprolactone.
The support for providing access to ScienceDirect and anti-plagiarism software from department of Research, Innovation and Consultancy, IKG Punjab Technical University, Jalandhar, is greatly acknowledged. The authors also wish to gratitude Chitkara University for providing facilities for this research.
Conflict of Interests
The authors report no conflicts of interest in this work.
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