New Validated Analytical Methods for the Determination of Methenamine Hippurate (An Antibiotic Agent)
Raghu Raj Naik*, Choppala Asha Deepti, Sistla Mounica Prathyusha, Mukthinuthalapati Mathrusri Annapurna
GITAM Institute of Pharmacy, GITAM (Deemed to be) University, Visakhapatnam-530045, India
*Corresponding Author E-mail: rajraghu496@gmail.com
ABSTRACT:
Methenamine hippurate is an antibiotic used for the treatment of urinary tract infections but no other types of bacterial infections (such as in the blood) or for viral infections (such as the common cold or flu). Methenamine hippurate acts as an antibiotic that stops the growth of bacteria in urine. Methenamine hippurate helps to make the urine acidic. When the urine is acidic, Methenamine hippurate turns into formaldehyde to kill the bacteria. Four new spectrophotometric methods have been proposed for the estimation of Methenamine hippurate in pharmaceutical formulations using acetate buffer (pH 2.4) and borate buffer (pH 9.0), phosphate buffer (pH 7.0) and phosphate buffer (pH 8.0). Methenamine hippurate has shown absorption maxima at 230 nm in all the methods. Methenamine hippurate obeys Beer-Lambert’s law over the concentration range 10-80 µg/ml in all the methods and all the methods were validated as per ICH guidelines.
KEYWORDS: Methenamine hippurate, Spectroscopy, Acetate buffer, Phosphate buffer, Borate buffer, Validation.
INTRODUCTION:
Methenamine or Hexamethylenetetramine or Hexamine or Urotropin (Figure 1) is a white crystalline compound is highly soluble in water and polar organic solvents. Methenamine a heterocyclic organic compound with a cage-like structure similar to adamantane. Methenamine hippurate is available as Methenamine hippurate and Methenamine mandelate. Methenamine hippurate is used for the treatment of urinary tract infection. Methenamine hippurate hydrolyzes to ammonia and formaldehyde (nonspecific antibacterial) in acidic urine. Mandelic acid or hippuric acid acidify the urine. Methenamine is chemically 1,3,5,7-tetraazatricyclo [3.3.1.13,7] decane. Methenamine hippurate has molecular formula C15H21N5O3 and molecular weight 319.37 gm and is also used for the production of plastic materials, explosives, phenolic resins, antibacterial pharmaceutics, disinfecting materials, in the rubber industry as an additive, and so forth1.
Methenamine was previously determined by ion-exchange HPLC2,
capillary gas chromatography3, spectrophotometry4-6, gas
chromatography7, solid phase spectrophotometry8,
fluorimetry9 and in biological samples such as urine by Proton NMR10
and ion pair extraction11, in edible animal tissues12 by
HPLC-MS/MS and in dairy products using GC-MS techniques13. In the
present study the authors have proposed new zero order and first order derivative
spectrophotometric methods for the determination of Methenamine hippurate using
four different buffers and all the methods were validated as per ICH
guidelines.
Figure 1: Chemical structure of Methenamine
MATERIALS AND METHODS:
Shimadzu double beam UV-visible spectrometer (model no. UV-1800) with quartz cells was used for the study and solutions were scanned at 200-400nm. Methenamine hippurate is available in brand names with name as HIPREX (Mylan Laboratories Ltd.) and UREX (Aurobindo Pharma) with labelled amount 1g. Buffer solutions such as acetate buffer pH 2.4 and borate buffer pH 9, phosphate buffer (pH 7.0 and pH 8.0) were prepared as per IP procedures. The stock solution of Methenamine hippurate was prepared by dissolving 25 mg of the drug in methanol in a 25 ml volumetric flask (1000µg/ml) and working standards were prepared by diluting the stock with respective buffer solutions such as acetate buffer, borate buffer and phosphate buffers.
Method validation
Zero order spectroscopy (D0)
A series of Methenamine hippurate solutions 10-80 µg/ml were prepared using different buffer solutions such as acetate buffer, borate buffer and phosphate buffers for Method A, B, C and D respectively and scanned against their reagent blank (200–400 nm). Methenamine hippurate has shown λmax at 230 nm in Method A, B, C and D (Figure 2). Calibration curves were drawn by taking the concentration on the X-axis and their respective absorbance on the Y-axis for Method A, B, C and D.
First order derivative spectroscopy (D1)
The individual zero order spectra of Methenamine hippurate obtained in above mentioned methods were converted into their first order derivative spectra with the help of inbuilt software of the instrument. The resultant derivative spectrum has shown minima at 244 nm for Method I and amplitude for Method II (219.31-244.33), Method III (219.00-245.00) and Method IV (219.00-245.67) (Figure 3). A calibration curve was constructed by using minima (Method I) or amplitude (Method II, III and IV) against concentration for all the methods. Precision was studied at three different concentration levels (n=3) and finally the percentage relative standard deviation was calculated whereas the accuracy studies were carried out by standard addition method.
Assay of Methenamine hippurate tablets
Twenty tablets of Methenamine hippurate were collected from two available brands and extracted with methanol. Dilutions were made after filtration with respective buffers and assay was carried out for Methods A, B, C and D in for zero order as well as first order techniques (Method I, II, III and IV).
RESULTS AND DISCUSSION:
New spectrophotometric techniques: zero order derivative (D0) and first order derivative (D1) were developed for the determination of Methenamine hippurate in different reagents such as acetate buffer pH 2.4 and borate buffer pH 9 phosphate buffer pH 7 and phosphate buffer pH 8 for methods A, B, C and D respectively.
Zero order spectroscopy (D0)
The absorption spectrum of Methenamine hippurate has shown (λmax 230 nm) in all the methods. Methenamine hippurate obeys Beer-Lambert’s law over the concentration range 10-80 µg/ml for all the methods (Table 1). Calibration curves were drawn by taking the concentration on the x-axis and the corresponding absorbance on the y-axis. The linear regression equations are found to be y = 0.032x + 0.005 (R˛ = 0.999), y = 0.034x + 0.008 (R˛ = 0.999), y = 0.034x + 0.008 (R˛ = 0.999), y = 0.034x - 0.008 (R˛ = 0.999) respectively for methods A, B, C, and D.
|
|
|
|
Method A |
Method B |
|
|
|
|
Method C |
Method D |
|
Figure 2: Absorption spectra of Methenamine hippurate (D0) |
|
Table 1: Linearity of Methenamine hippurate – Zero derivative spectroscopy
|
Conc. (µg/ml) |
Absorbance |
|||
|
Method A |
Method B |
Method C |
Method D |
|
|
10 |
0.348 |
0.365 |
0.341 |
0.336 |
|
20 |
0.637 |
0.694 |
0.694 |
0.663 |
|
30 |
0.965 |
1.047 |
1.049 |
1.02 |
|
40 |
1.335 |
1.363 |
1.43 |
1.396 |
|
50 |
1.611 |
1.729 |
1.81 |
1.713 |
|
60 |
1.969 |
2.074 |
2.203 |
2.05 |
|
80 |
2.596 |
2.754 |
2.95 |
2.755 |
First order derivative spectroscopy (D1)
Methenamine hippurate obeys Beer-Lambert’s law over the concentration range 10-80 µg/ml in all the methods (Table 2). The linear regression equations are found to be y = 0.001x + 0.000 (R˛ = 0.999), y = 0.002x + 0.000 (R˛ = 0.999), y = 0.002x - 0.000 (R˛ = 0.999), y = 0.002x - 0.000 (R˛ = 0.999) respectively for Method I, II, III and IV.
|
|
|
|
Method I |
Method II |
|
|
|
|
Method III |
Method IV |
|
Figure 3: Overlay first derivative spectra of Methenamine hippurate (D1) |
|
Table 2: Linearity of Methenamine hippurate – First derivative spectroscopy (Max: Maxima; Min: Minima)
|
Conc. (µg/ml) |
Method I |
Method II |
Method III |
Method IV |
||||||
|
Min |
Max |
Min |
Amplitude |
Max |
Min |
Amplitude |
Max |
Min |
Amplitude |
|
|
10 |
0.014 |
0.009 |
0.013 |
0.022 |
0.013 |
0.009 |
0.022 |
0.013 |
0.009 |
0.022 |
|
20 |
0.029 |
0.017 |
0.027 |
0.044 |
0.029 |
0.018 |
0.047 |
0.026 |
0.018 |
0.043 |
|
30 |
0.042 |
0.026 |
0.041 |
0.067 |
0.041 |
0.026 |
0.067 |
0.041 |
0.027 |
0.067 |
|
40 |
0.056 |
0.034 |
0.054 |
0.088 |
0.050 |
0.035 |
0.091 |
0.053 |
0.035 |
0.088 |
|
50 |
0.070 |
0.042 |
0.067 |
0.109 |
0.071 |
0.044 |
0.115 |
0.067 |
0.044 |
0.111 |
|
60 |
0.084 |
0.050 |
0.081 |
0.131 |
0.083 |
0.054 |
0.137 |
0.080 |
0.053 |
0.133 |
|
80 |
0.111 |
0.066 |
0.107 |
0.175 |
0.112 |
0.071 |
0.183 |
0.107 |
0.071 |
0.178 |
Method validation and Assay of Methenamine hippurate
The RSD in precision (Table 3) and accuracy (Table 4) studies for both zero order and first derivative spectral methods were tabulated and found to be < 2 indicating that the methods are precise and accurate. The percentage of purity of Methenamine hippurate was found to be greater than 99 % in all the methods and the methods can be applied for the quantitative determination of pharmaceutical formulations (Table 5). The Optical characteristics of Methenamine hippurate were shown in Table 6.
Table 3: Precision studies of Methenamine hippurate
|
Zero order spectroscopy |
||||
|
Conc.(µg/ml) |
Intraday precision: Mean ± SD ( RSD) |
|||
|
Method A |
Method B |
Method C |
Method D |
|
|
10 |
0.432±0.0015 (0.347) |
0.361±0.0015 (0.415) |
0.335±0.003 (0.895) |
0.336±0.002 (0.531) |
|
30 |
0.959±0.0032 (0.333) |
1.042±0.0032 (0.335) |
1.043±0.004 (0.383) |
1.018±0.0035 (0.309) |
|
60 |
1.961±0.0032 (0.163) |
2.041±0.0023 (0.121) |
2.199±0.004 (0.909) |
2.08±0.003 (0.143) |
|
Conc.(µg/ml) |
Inter day precision: Mean ± SD ( RSD) |
|||
|
|
Method A |
Method B |
Method C |
Method D |
|
10 |
0.437±0.0015 (0.343) |
0.365±0.0015 (0.410) |
0.338±0.0003 (0.880 ) |
0.334±0.002 (0.598) |
|
30 |
0.965±0.0032 (0.331) |
1.047±0.0035 (0.334) |
1.045±0.0004 (0.382) |
1.012±0.006 (0.594) |
|
60 |
1.969±0.0032 (0.162) |
2.047±0.0025 (0.122) |
2.201±0.0002 (0.908) |
2.05±0.015 (0.731) |
|
First derivative spectroscopy |
||||
|
Intraday precision: Mean ± SD ( RSD) |
||||
|
Conc. µg/ml) |
Method I |
Method II |
Method III |
Method IV |
|
10 |
0.014±0.0001 (0.833) |
0.018±0.00015 (0.833) |
0.026±0.00015 (0.517) |
0.021±0.0001 (0.588) |
|
30 |
0.038±0.0003 (0.967) |
0.062±0.0003 (0.447) |
0.059±0.00015 (0.254) |
0.061±0.0002 (0.645) |
|
60 |
0.079±0.0003 (0.379) |
0.129±0.0003 (0.232) |
0.117±0.0003 (0.254) |
0.129±0.0002 (0.188) |
|
Inter day precision: Mean ± SD ( RSD) |
||||
|
Conc. µg/ml) |
Method I |
Method II |
Method III |
Method IV |
|
10 |
0.014±0.0001 (0.714) |
0.022±0.00015 (0.681) |
0.022±0.0001 (0.681) |
0.022±0.0001 (0.526) |
|
30 |
0.042±0.0003 (0.714) |
0.067±0.0003 (0.447) |
0.067±0.0001 (0.238) |
0.068±0.0002 (0.625) |
|
60 |
0.084±0.0003 (0.357) |
0.131±0.0003 (0.229) |
0.137±0.0003 (0.236) |
0.133±0.0002 (0.183) |
Table 4: Accuracy studies of Methenamine hippurate
|
Zero order spectroscopy |
||||||
|
Spiked Conc. |
Formu lation |
Total Conc. |
Conc. obtained (μg/ml) (RSD) Recovery |
|||
|
Method A |
Method B |
Method C |
Method D |
|||
|
10 (50 %) |
20 |
30 |
29.82 (0.003) 99.4 |
29.23 (0.005) 97.4 |
29.65 (0.011) 98.8 |
29.86 (0.005) 99.5 |
|
20 (100 %) |
20 |
40 |
39.52 (0.004) 98.8 |
39.28 (0.003) 98.45 |
39.23 (0.008) 98.00 |
39.87 (0.004) 99.6 |
|
30 (150 %) |
20 |
50 |
49.67 (0.002) 99.34 |
49.69 (0.003) 99.3 |
49.73 (0.006) 99.4 |
49.83 (0.003) 99.6 |
|
First derivative spectroscopy |
||||||
|
Spiked Conc. |
Formu lation |
Total Conc. |
Conc. obtained (μg/ml) (RSD) Recovery |
|
||
|
Method I |
Method II |
Method III |
Method IV |
|||
|
10 (50 %) |
20 |
30 |
29.72 (0.004) 99.06 |
29.61 (0.003) 98.7 |
29.23 (0.011) 97.4 |
29.36 (0.011) 97.8 |
|
20 (100 %) |
20 |
40 |
39.82 (0.004) 99.5 |
39.22 (0.006) 98.00 |
39.72 (0.008)\ 99.3 |
39.29 (0.008) 98.2 |
|
30 (150 %) |
20 |
50 |
49.68 (0.003) 99.3 |
49.11 (0.003) 98.2 |
49.68 (0.006) 99.3 |
49.52 (0.006) 99.04 |
Table 5: Assay of Methenamine hippurate (Label claim: 1 gm)
|
Zero order derivative spectroscopy |
||||||||
|
Brand |
Method A |
Method B |
Method C |
Method D |
||||
|
Observed amount (gm) |
Recovery |
Observed amount (gm) |
Recovery |
Observed amount (gm) |
Recovery |
Observed Amount (gm) |
Recovery |
|
|
I |
0.9988 |
99.88 |
0.9933 |
99.33 |
0.9982 |
99.82 |
0.9942 |
99.42 |
|
II |
0.9979 |
99.79 |
0.9981 |
99.81 |
0.9987 |
99.87 |
0.9993 |
99.93 |
|
First order derivative spectroscopy |
||||||||
|
Brand |
Method I |
Method II |
Method III |
Method IV |
||||
|
Observed amount (gm) |
Recovery |
Observed amount (gm) |
Recovery |
Observed amount (gm) |
Recovery |
Observed Amount (gm) |
Recovery |
|
|
I |
0.9974 |
99.74 |
0.9919 |
99.19 |
0.9928 |
99.28 |
0.9931 |
99.31 |
|
II |
0.9961 |
99.61 |
0.9931 |
99.31 |
0.9949 |
99.49 |
0.9967 |
99.67 |
Table 6: Optical characteristics of Methenamine hippurate – Zero order spectroscopy
|
Parameters |
Method A |
B |
C |
D |
||
|
Linearity (µg/ml) |
10-80 |
10-80 |
10-80 |
10-80 |
||
|
λmax (nm) |
230 |
230 |
230 |
230 |
||
|
Molar extinction coefficient (litre/ mole/ cm-1) |
1.11×104 |
1.16×104 |
1.08×104 |
1.07×104 |
||
|
Sandell’s sensitivity (µg/cm2/0.001 absorbance unit) |
0.287 |
0.273 |
0.029 |
0.029 |
||
|
Slope |
0.034 |
0.037 |
0.034 |
0.034 |
||
|
Intercept |
0.005 |
0.008 |
0.030 |
0.008 |
||
|
Correlation coefficient |
0.9999 |
0.9993 |
0.9996 |
0.9999 |
||
|
Precision (RSD) |
Intraday |
0.13-0.38 |
0.15-0.29 |
0.15-0.32 |
0.19-0.37 |
|
|
Inter day |
0.39-0.56 |
0.38-0.46 |
0.39-0.64 |
0.43-0.53 |
|
|
Accuracy ( RSD) |
0.38-0.56 |
0.15-0.38 |
0.25-0.45 |
0.73-0.92 |
||
CONCLUSION:
The new spectrophotometric methods were validated for the determination of Methenamine hippurate and found to be simple, precise and accurate and the methods can be successfully applied for the determination of Methenamine hippurate in pharmaceutical dosage forms.
ACKNOWLEDGMENT:
The authors are grateful to M/s GITAM (Deemed to be University), Visakhapatnam for providing the research facilities and to Mylan Laboratories for supplying gift samples of Methenamine hippurate.
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Received on 10.01.2020 Modified on 22.02.2020
Accepted on 03.03.2020 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(3):1386-1390.
DOI: 10.5958/0974-360X.2020.00254.1