Colorimetric Determination of Sulfadiazine in Tablets with para-dimethylaminobenzaldehyde (p -DMAB) -DMAB) Usi Using ng External Standard Calibr ation Techni ques Mariel Lyka S. Co, Maria Kristina E. Frogoso, Merrene Bright D. Judan*, Airielle Janine M. Ordoñez, Bianca Kamille Takata, Amanda Beiya Karina P. Tejano Pharmaceutical Chemistry Department, College of Pharmacy, Valenzuela Hall, University of the Philippines Manila, Taft Avenue, Ermita, Manila 1000
*Corresponding author:
[email protected]
ABSTRACT AB STRACT In this experiment, the amount of sulfadiazine in tablets was determined through colorimetric method using external standard calibration. Sulfadiazine was reacted with pdimethylaminobenzaldehyde (p-DMAB), which resulted to a yellow-colored solution. Its absorbance was measured using a spectrophotometer at 450 nm. The calculated label claim was 214.32% for the single-standard method and 209.50% for multiple-standard method. The sample did not conform to the official requirement of not less than 95.0% and not more than 105.0% of the labeled amount of sulfadiazine; however, the procedure employed is not the one indicated in the USP monograph.
Keywords : colorimetry, sulfadiazine, p-dimethylaminobenzaldehyde, external standard
calibration
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I. INTRODUCTION
Molecular absorption spectrophotometry is a technique used to measure the absorption of electromagnetic radiation in its passage through a gas, a liquid, or a solid in the UV-Vis regions. It is widely used for structural elucidation of various substances as it is also more beneficial since a very small quantity of substance is required for analysis (Fayed, n.d.). Measuring the light absorbed by the molecules a certain solution is governed by the Beer-Lambert Law: A=εbc
(Equation 1)
which states that when light passes through a solution, its absorbance is directly proportional to the path length of the absorbing medium ( b); the molar concentration (c) of the species being examined (Skoog, et al., 2014); and the molar absorptivity of the substance ( ε ). Colorimetry is the measurement of the amount of light absorbed by the color developed in a sample solution. It is measured in the visible region of the spectrum (350-900 nm), which provides many advantages for quantitative determination because of its selectivity. Samples used should be hi ghly colored or could be reacted with a chromogenic agent. This method was performed in this experiment to quantify the amount of sulfadiazine present in the weighed powdered tablets using the single a nd multiple standard external calibration techniques. Sulfadiazine (C10H10N4O2S) is a sulfonamide antibiotic with a wide spectrum against most grampositive and many gram-negative organisms (Drugs.com, 2017). It was reacted with the chromogenic agent, para-dimethylaminobenzaldehyde (p-DMAB), to form a yellow-colored imine product which could be measured in the visible region due to the presence of a chromophore. Sulfadiazine tablets (C10H10N4O2S) contains not less than 95.0% and not more than 105.0% of the labelled amount of C10H10N4O2S (USP 35, 2012). Each tablet contains 125 mg sulfadiazine. The aim of this experiment is to determine the amount of sulfadiazine in tablets, percentage label claim, ʎmax, molar absorptivity, linear range, limit of detection (LOD), limit of quantitation (LOQ), and recovery of sulfadiazine from the tablet matrix.
II. METHODS
Single Standard Method Standard preparation. To 10 mL of 1N hydrochloric acid, 25 mg of previously dried sulfadiazine
standard was added. The mixture was heated and stirred occasionally until the powder is completely dissolved. It was, then, cooled to room temperature, transferred to a 25-mL volumetric flask and was brought to volume using 1N hydrochloric acid. From the the resulting mixture which served as the standard stock solution, 1.25 mL was transferred to a 25 -mL volumetric flask with 5 mL of p-DMAB and was brought to volume using 1N hydrochloric acid. The final solution was, then, triplicated. Assay preparation. The average weight of previously weighed and powdered 20 sulfadiazine
tablets was determined. A weight equivalent to 25 mg of sulfadiazine was added to 10 mL of 1N hydrochloric
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acid. The mixture was heated and occasionally stirred for about 5 minutes. It was cooled to room temperature and was filtered. The filtrate was transferred to a 25-mL volumetric flask and was brought to volume using 1N hydrochloric acid. From the resulting mixture, 1.25 mL was transferred to a 25-mL volumetric flask with 5 mL of p-DMAB and was brought to volume using 1N hydrochloric acid. The final solution was, then, triplicated. Blank preparation. The blank preparation was prepared in a 25-mL volumetric flask using 5 mL of p-DMAB that was brought to volume using 1N hydrochloric acid. Determination of wavelength. The absorbance of the Standard stock solution was measured
against a blank. Using the scanning feature of the spectrophotometer, the wavelength of maximum absorption from 480 nm to 230 nm was determined. This wavelength was used in the subsequent measurements. Determination of the Absorption Spectrum of Sulfadiazine. Using the underivatized form of the
preparation, the same procedure as the determination of wavelength was performed. Assay. The absorbance of the Standard preparation and the Assay preparation was measured
against a blank. The percentage label claim was determined.
Multiple Standard Method Standard preparation. The standard preparation involved the same procedure as that of the single
standard method. However, instead of using a single standard of 2 5 mg sulfadiazine, four standards were accurately weighed: 10 mg, 20 mg, 40 mg, 80 mg, and 160 mg of sulfadiazine. Each sulfadiazine standard was transferred to individual 25-mL volumetric flasks. Assay preparation. The assay preparation obtained from the single standard method was used. Assay. The absorbance of the solutions from the Standard preparation and Assay preparation was
obtained from the spectrophotometer at a determined wavelength. Three absorbance readings were obtained from each solution and the average value was calculated. Then, a standard curve was constructed by plotting the absorbance (y-axis) against the concentration (x-axis) of the sulfadiazine standards. Using the least square method, the best fit line was determined. The concentration of the Assay preparation was interpolated from the equation of the best fit line. Then, the amount of sulfadiazine in the tablet (mg), percentage label claim, molar absorptivity, limit of detection (LOD), and limit of quantitation (LOQ) were determined.
Hazards The hazardous reagents employed in the experiment were hydrochloric acid and p-DMAB. Use the fume hood when handling such chemicals (Bermudez, Chua, & Racho, 2016). Hydrochloric acid is known for its white pungent fumes especially when concentrated. The vapors are irritants of the respiratory system, eyes, and skin and they are known to cause severe irritation, chemical and inhalational burns. Prevent
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contact with skin and avoid breathing vapor. Disposal is done by adding calcium carbonate until neutralization is complete: 2 HCl + Na2CO3 → 2 NaCl + CO2 + H2O
Treat any solid residue remaining as a normal refuse (Armour, 2003). p-DMAB, on the other hand, is an irritant of the skin and eyes, and is severely hazardous when
ingested. The compound is toxic to the mucous membranes in the lungs. Avoid co ntact as it can permeate the skin. Repeated or prolonged exposure can cause target organ damage (Owen, 2003). Dispose the chemical in the halogenated waste bottle (Bermudez, Chua, & Racho, 2016).
III. RESULTS
Tables 1 to 3 summarize the weighing and absorbance data of the methods employed in the experiment. The wavelength with the highest value of absorbance (Vmax) is used in the analysis of the sulfadiazine tablets, which is found to be at 450 nm. The sulfadiazine used in the experiment has a label claim of 250 mg per tablet. On the average, each tablet weighs 0.0758 g.
Table 1 Absorbance Readings obtained from Single-Standard Method using a Standard Solution of Sulfadiazene (SDZ) Trial
Wt. of SDZ
Final Working SDZ
Standard (mg)
Concentration (mg/mL)
1
26
0.052
0.334
2
26
0.052
0.316
3
26
0.052
0.316
Mean±SD
26±0.0000
0.052±0.0000
0.322±0.008485
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Ab so rb anc e
Table 2
Absorbance Readings obtained from Multiple -Standard Method using Standard Solutions of Sulfadiazine (SDZ) Wt. of SDZ
Final Working SDZ
Standard
Concentration
(mg)
(mg/mL)
1
2
3
1
10.8
0.0216
0.140
0.144
0.146
2
20.5
0.0410
0.244
0.244
0.231
3
40.0
0.0800
0.493
0.494
0.493
4
79.8
0.1596
0.985
0.961
0.982
5
161.1
0.3222
2.107
2.100
2.101
Solution
Ab so rb anc e Mean±SD
0.1433 ± 0.002494 0.2396 ± 0.006128 0.4933 ± 0.000471 0.9760 ± 0.010678 2.1027 ± 0.003091
Table 3 Absorbance Readings obtained from the Sulfadia zine Assay Preparation Trial
Wt. of SDZ
Final Working SDZ
Ab so rb anc e
Standard (mg)
Concentration (mg/mL)
1
75.8
0.1516
0.763
2
75.8
0.1516
0.742
3
75.8
0.1516
0.502
Mean±SD
75.8±0.0000
0.1516±0.0000
0.669±0.1183976
Figure 1 shows the standard curve of sulfadiazine (R 2 = 0.9989). Figures 2 and 3 show the absorption spectra of the underivatized and derivatized sulfadiazine, respectively.
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2.5
y = 6.5072x - 0.018 R² = 0.9989
2
e c n a b r o s b A
1.5
1
0.5
0 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
-0.5
Concentration (mg/mL) Figure 1. Calibration data from multiple standard method using standard solutions of Sulfadiazine
Figure 2. Absorption spectra of underivatized sulfadiazine
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Figure 3. Absorption spectra of derivatized sulfadiazine
IV. DISCUSSION
Sulfadiazine, the sample for this experiment, is colorless in solution. Therefore, its colorimetric determination can only be achieved through the addition of a chromogenic agent. Paradimethylaminobenzaldehyde (p-DMAB) was used, which reacts with primary amines to produce highly colored substances (Sadek, 2004). Through Schiff base formation, p-DMAB and sulfadiazine form a yellow imine product whose absorbance can be mea sured at around 450-480 nm (Knevel & DiGangi, 1977). CH3 N H3C
H2N
H3C
N
O O
N H3C
N
O
S
+
O
N
S
NH
N
→
O
NH
N
Figure 4. Reaction of p-DMAB and sulfadiazine forming a highly colored yellow imine product
In the assay of sulfadiazine tablets, the absorbance spectra of sulfadiazine were determined with and without p-DMAB. This is to show that the absorbance was due to the reaction of sulfadiazine with pDMAB. The maximum absorption was found in the visible region due to its better sensitivity as compared with measuring in the UV region. T he maximum absorbance measured was 450 nm as shown in Figure 2. For the single standard method, the average absorbance of a 0.052 mg/mL -sulfadiazine standard is 0.322. The average distance of each observation from the average absorbance is 0.0085. This in turn, means that a high degree of precision was observed during the measurement of the standard. Other computed parameters are shown in Table 4.
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For the multiple standard method, linear regression analysis was performed with mean absorbance versus the final working concentration of sulfadiazine (in mg/mL). The equation of the line y = 6.5072 x 0.018 was obtained with R 2 = 0.9989. The R 2 value approaches the value of 1, which indicates a strong, direct, linear relationship among the data. Other computed parameters are shown in Table 4. Based on the percentage label claim computed using single and multiple standard method, the sample did not conform to the official requirement stated in the USP.
Table 4
Computed values for the Single-Standard and Multiple-Standard Method
Amount of sulfadiazine per tablet (mg) Percentage label claim (%) ʎmax (nm) Molar absorptivity (L/mol·cm) Linear range (mg/mL) LOD LOQ
Single-Standard Method 54 214.32 450 1549.81 N/A N/A N/A
Multiple-Standard Method 52.79 209.50 450 1585.88 0.0000 – 3.222 N/A N/A
The linear range is 0.0000 mg/mL to 0.3222 mg/mL. The limits of quantitation and limits of detection were not computed since the instrument already accounted for the absorbance of the blank.
V. CONCLUSION
The sulfadiazine in tablets was derivatized for colorimetric determination using external standard calibration. Determination was done at 450 nm. However, the calculated am ount of sulfadiazine present in the sample did not conform to the USP standards. Based on the results, the multiple standard yielded a more precise determination. It also showed a strong, direct, linear relatio nship between concentration and absorption. The limits of quantitation and limits of detection were not computed since the instrument already accounted for the absorbance of the blank. It is recommended to use more replicates of each solution (at least five replicates) and use m ore standards for the multiple standard method.
VI. LITERATURE CITED
Armour, M. A. (2003). Hazardous laboratory chemicals disposal guide (3rd ed.). Boca Raton, FL: CRC Press LLC. Bermudez, D. M., Chua, C. C., & Racho, M. C. P. (2016). A handbook of good laboratory practices for faculty, students, researches, and laboratory personnel . Ermita: MNL: College of Pharmacy, University of the Philippines Manila. Drugs.com. (2017). Sulfadiazine 500 mg tablets, BP. Retrieved, February 6, 2018 from http://www.drugs.com/uk/sulfadiazine-500mg-tablets-bp-leaflet.html
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Fayed, T. (n.d.). UV-Vis molecular absorption spectroscopy. Retrieved, February 11, 2018 from http://sci.tanta.edu.eg/files/UV-Vis%20molecular%20absorption%20spectroscopy-%20BScLect%205.pdf Knevel, A.M. & DiGangi, F.E. (1977). McGraw-Hill.
Jenkins’
quantitative pharmaceutical chemistry (7th ed.). New York:
Owen, S. (2003) Material safety data sheet: P-dimethylaminobenzaldehyde. Retrieved, February 10, 2018, from https://www.lewisu.edu/academics/biology/pdf/p-Dimethylaminobenzaldehyde.pdf Sadek, P.C. (2004). Illustrated pocket dictionary of chromatography. NY: John Wiley & Sons. Skoog, D. A., West, D. M., Holler, F.J., & Crouch, S.R. (2014). Fundamentals of analytical chemistry (9th ed.). Belmont, CA: Brooks/Cole.
VII. APPENDIX
Computations A. Si ng le-Stan dar d Meth od A=εbC 0.322 = ε x 1 cm x 0.052 mg/mL ε = 6.1923 mL∙mg-1/cm-1 A=εbC 0.669 = 6.1923 mL∙mg-1/cm-1 x 1 cm x C C = 0.1080 mg/mL x 500 mL C = 54 mg
%AI=(computed weight of SDZ tab)/(initial weight of SDZ tab) x 1 00% = (54 mg)/(75.8 mg) x 100% = 71.24% % LC= (% AI x ave.weight of 20 tabs)/(labeled claim) = (71.24% x 0.7521 g)/(0.25 g/tab) = 214.32%
Molar Absorptivity A = ɛbc ɛ = A/bc
ɛ=0.322/(1 cm) 0.052 g/L⁄250.28 / ɛ = 1549.81 L/mol·cm
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B. Multipl e Standard Method
Computation: y= 6.5072x - 0.018 R2=0.9988 0.669 = 6.5072x - 0.018 x= 0.10558 mg/mL 0.10558 mg/mL x 500 mL= 52.79 mg
%AI=(computed weight of SDZ tab)/(initial weight of SDZ tab) x 100% = (52.79 mg)/(75.8 mg) x 100% = 69.64% % LC= (% AI x ave.weight of 20 tabs)/(labeled claim) = (69.64% x 0.7521 g)/(0.25 g/tab) = 209.50%
Molar Absorptivity A = ɛbc ɛ = A/bc
ɛ=0.669/(1 cm) 0.10558 g/L⁄250.28 / ɛ = 1585.88 L/mol·cm
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