The Drug Substance
Analysis of Sulfonamides
Abstraction
In the scientific discipline of rational drug design, log P value of a compound is of import to find its extent of capableness to go through through cell membrane. In finding log P of sulfa drugs, Thin-layer Chromatography ( TLC ) , ‘shake-flask ‘ method and High Performance Liquid Chromatography ( HPLC ) were used. Chemical pureness of an unknown sulfa drug, C26 was determined utilizing HPLC. Straight stage TLC is non suited in determine the divider coefficient of sulphonamides due to the ground it is unable to associate its divider and keeping factor. HPLC was found to be the best method in finding log P as it is a dependable and accurate method widely used in pharmaceutical industries. The Ptrue values obtained for sulfathiazole in pH 1 and pH9 utilizing ‘shake-flask ‘ method were 1.47 and 3.1859 severally. The log P value obtained from HPLC for C26 is 3.300 and chemical pureness value was 68 % .
Keywords:
log P, sulfa drugs, TLC, ‘shake-flask ‘ , HPLC
Introduction
Sulfa drugs are antibacterial drugs used to handle infections caused by micro-organisms. Sulfonamide antibiotics mark on enzyme dihydropteroate synthase ( DHPS ) which plays an of import function in catalyzing the bacterial production. They work by suppress the activity of DHPS which finally inhibits the synthesis of folic acid and DNA ( Foye et al, 1995 ) . Sulfonamides imitate p-aminobenzoic acid ( PABA ) and vie for the active site. In this manner, bacteriums could non multiply as there are insufficient of vitamin Bcs available for their growing. In fabricating the needed sulfa drug ( C26 ) , an aminoalkane which is known as 4-aminoacetophenone is required to obtain the coveted concluding compound.
Sulfonamides ‘ comparative distribution in an n-octanol/water mixture can be done by experimentation by mensurating the hydrophobic character of the compound. Partition coefficient, P is one of the most important factors in commanding the drug action in biological systems. The divider coefficient is an of import step in foretelling the soaking up, distribution and riddance of drugs in the organic structure. Not merely that, it can be used to associate the biological activity of a drug to it belongingss. Log P, which is known as octanol-water divider coefficient, is used in rational drug design to mensurate the hydrophobicity of a compound.
It is of import to prove the hydrophobic character of a drug as it will impact the extent the drug crosses cell membranes easy. The different substituents on a drug have different hydrophobic character and finally hold different biological activity ( Patrick 2009 ) . Hydrophobic compounds have a high P value and frailty versa for hydrophilic compounds.
Log P can be measured with different methods. Namely, ‘shake-flask ‘ method, thin-layer chromatography, and High Partition Liquid Chromatography ( HPLC ) can be used to mensurate log P. HPLC and TLC are known to be giving a hydrophobicity parametric quantity instead than a divider coefficient ( Takacs-Novak and Avdeef, 1996 ) .
The intent of this survey was to look into the belongingss of sulphonamides, particularly the hydrophobicity of the compounds. Log P was determined utilizing assorted methods and the advantages and disadvantages of each method used were investigated. Chemical pureness of the compound was investigated utilizing the modern HPLC system.
Experimental
Establishing the log P of sulfa drugs utilizing TLC
In the separation of sulfa drugs, TLC was used to set up the log P of the sulphonamides. Sulfonamide solutions were prepared by fade outing 40mg of sulfa drug in propanone and the solutions were made up to 10mL in a volumetric flask which will give a concentration of 0.4 % w/v. Then, each single sulfa drug solution was transferred to a glass capillary tubing. After that, the solutions contained in the glass capillary tubing were spotted on a home base with silicon oxide coated and aluminum backed size at 12cm ? 7cm. The home base was so placed in a chromatographic chamber incorporating nomadic stage butan-1-ol for about 60 proceedingss. When 75 % of the nomadic stage moves up the home base, the home base was removed from the chromatographic chamber and the nomadic stage forepart was marked. Hot air was washed to dry the home base and the sulfa drugs were visualized under short moving ridge UV and the centre of each detected topographic point was marked. The distances from the musca volitanss to the nomadic stage forepart and to the centre of the detected topographic point of each sulfa drugs were measured utilizing a swayer. The keeping factor, Rf value for each sulfa drugs was so calculated by following expression:
Rf = Distance moved by topographic point
Distance moved by dissolver
Researching the breakdown of sulfa drug under ionizing and non-ionising conditions utilizing the ‘shake-flask ‘ method
A 25µg mL-1 solution in 0.5M NaOH ( Solution A ) and 10µg mL-1 solution in H2O ( Solution B ) were prepared utilizing a stock solution incorporating 0.02 % w/v of sulfa drug in H2O. A scope of standardization criterions which contain 2.3, 5, 7.5,10, 12.5, and 15µg mL-1 of sulfa drug in 0.5M NaOH was prepared utilizing Solution A. The maximal optical density of 1.0774 was determined at the wavelength of 257nm utilizing the 15µg mL-1 criterion. The criterions ‘ optical density was read at the ?max of 1.0774 with NaOH as the space. A graph of optical density versus concentration for the sulfa drug was plotted utilizing the readings.
Two partitioning samples were prepared by adding 10ml of Solution B, 10mL 0.1M HCl and 20mL octanol in dividing funnel ( I ) and 10mL of Solution B, 10mL pH 9.0 buffer and 20mL octanol in dividing funnel ( two ) . The funnels were shaken at frequent intervals for 30 proceedingss so that the beds were separated to the full. It is of import to do certain the funnels were non shaken excessively smartly to avoid an emulsion to be formed. Then, the aqueous bed was carefully run away and 20mL 0.5M NaOH was added utilizing a pipette to the staying octanol in the funnel. The funnel was shaken once more for 5 proceedingss for separation and the optical density of the aqueous bed was measured at the ?max of 1.0774. The concentration of the sulfa drugs in the 0.5M NaOH was calculated from the standardization curve.
Determination of log P values utilizing HPLC
The sample solution was injected into HPLC ( Agilent 1120 Compact LC ) with column Zorbax C8 measured at 150 millimeters X 4.6 mm ID. The wavelength is set at 254 nanometer, flow rate at 1 mL min-1, temperature at 40oC, and injection volume of 20 µL. The running clip of the system is set to be 120 proceedingss. The ratio of nomadic stage used in system is 85:15 ( 0.1 % CH3COOH: MeOH ) % v/v.
Chemical Purity Determination
A sample solution of C26 at a concentration of 0.1 mg mL-1 was prepared in a 100mL volumetric flask. 7.2mL of methyl alcohol was added to fade out the sample and nomadic stage was added to do up to 100mL. 20µL of the sample solution was injected to the HPLC-DAD system with column C18 Phenomenex Luna 5µ measured at 150 millimeters X 4.6 millimeters ID utilizing a individual injection manner and the method ‘Chempur 1 ‘ . The flow rate of the system was set at 1 mL min-1. The ratio of the nomadic stage used in the HPLC system is 85:15 ( CH3COOH: MeOH ) % v/v.
Consequences and Discussion
Establishing the log P of sulfa drugs utilizing thin-layer chromatography
Sulfa drugs
Log Papp
Log Ptrue
Releasing factor
Log Rf
Sulfacetamide
-1.0
-1
0.6
-0.2218
Sulfadiazine
-1.3 at pH 7.5
-0.2586
0.5333
-0.273
Sulfamethoxazole
0.9
0.9
0.88
-0.05552
Sulfanilamide
-0.6
-0.6
0.6533
-0.18487
Sulfapyridine
0.4
0.4
0.4933
-0.30686
Sulfathiazole
-0.4 at pH 7.5
0.14554
0.5333
-0.273
Table 1: Log P and Log Rf values of sulfa drugs
The literature values for log P of the sulphonamides were listed in the tabular array above. A graph log Rf versus log P was drawn to compare gauge the extent of partitioning against the literature values of log P. Based on Figure 3, it was noticed that there was no relationship between log Rf and log P as the arrested development coefficient, R2 value was 0.1017. This little value of R2 of 0.1017 shows that merely 10.17 % of fluctuation in biological activity was accounted for by the parametric quantities of log P and log Rf ( Patrick, 2009 ) . However, R value should non be relied on excessively much as the value obtained does non take any history of the figure of compounds that were involved in the survey. This means that a higher value of R2 could be obtained if there were more compounds incorporated in the survey.
This could be due to consecutive stage TLC was used in the experiment. The stationary stage in the experiment is the silicon oxide bed which will interact with hydrophilicity of a compound. Sulfa drugs are hydrophobic compounds so it is best to use a stationary stage which will interact with the hydrophobicity belongings of a compound. To better the consequences, reversed stage TLC should be considered in finding log P value which is the chief concern of hydrophobicity of a compound. In reversed stage TLC, the silicon oxide home base has increased sum of alkyl groups which will enable hydrophobic-hydrophobic interaction between the stationary stage and the compound. It will besides increase the clip of the compound traveling in the nomadic stage.
The advantage of utilizing TLC is that log P of many compounds can be determined on one home base. Small sum of sample is required to undergo TLC procedure. This method is cheaper compared to other methods in finding log P of the compounds. TLC besides has the advantage that all components of a sample can be visualised easy, particularly under UV visible radiation. In contrast, the quantitation from the musca volitanss obtained from the home base is non easy as TLC is more suited for the separation of involatile compounds.
Researching the breakdown of sulphonamide under ionizing and non-ionising conditions utilizing the ‘shake-flask ‘ method
In shake-flask method, the pKa values are required to cipher the divider coefficient ( Takacs-Novak and Avdeef, 1996 ) .
Calibration criterions for sulfathiazole:
Concentration ( µg mol-1 )
Optical density 1
Optical density 2
Average optical density
0.0
0.000
0.000
0.000
2.5
0.222
0.221
0.222
5.0
0.404
0.404
0.404
7.5
0.567
0.555
0.566
10.0
0.737
0.737
0.737
12.5
0.920
0.920
0.92
15.0
1.073
1.075
1.074
Table 2: Concentrations and mean optical density for the standardization criterions of sulfathiazole
Partitioning samples:
Funnels
pH
Optical density 1
Optical density 2
Average optical density
I
1
0.219
0.218
0.219
two
9
0.014
0.014
0.014
Table 3: Optical densities obtained in different pH.
From Figure 4, the equation obtained from the additive line is y=0.0735x.
The optical density value obtained for Funnel ( I ) is 0.219. The value was substituted in the equation as Y to obtain x value which is the concentration of sulfathiazole for in 0.5M NaOH.
For Funnel ( I ) ;
Y = 0.0735x
0.219 = 0.0735x
ten = 2.9796 µg mL-1
For Funnel ( two ) ;
0.014 = 0.0735x
ten = 0.1905 µg mL-1
Funnels
Optical density
Concentration ( µg mL-1 )
pH
Entire sulfa drug sum in organic stage ( µg )
Entire sulfa drug sum in aqueous stage ( µg )
Papp
Ptrue
I
0.219
2.9796
1
59.592
40.408
1.47
1.47
two
0.014
0.1905
9
3.810
96.19
0.039609
3.1859
Table 4: Calculated Ptrue obtained from the concentration in different pH.
To cipher Ptrue from the concentration of the sulfa drug contained in 0.5M NaOH, entire sum of drug in organic and aqueous stage, Papp and pH of the buffer are required. The on the job computations are as below:
For Funnel ( I ) in pH 1 ;
Concentration of sulfa drug in organic stage = 2.9796 µg mL-1
1 milliliter of the organic stage contained 2.9796 µg of drug, this means that 20 milliliter of organic stage contained a sum of 59.592 µg of sulfa drug.
Entire sulfa drug in organic and aqueous stage = 10 milliliter X 10 µg mL-1
= 100 µg
Entire sulfa drug in aqueous stage = 100 µg – 59.592 µg
= 40.408 µg
Papp = [ sulfa drug ] in organic stage / [ sulfa drug ] in aqueous stage
= ( 59.592/20 ) / ( 40.408/20 )
= 1.47
In this instance, Papp depends on pH of the solution.
log D at pH 1 = log P – log [ 1 + 10 ( pH-pKa ) ]
log Ptrue = log 1.47 + log [ 1 + 10 ( 1-7.1 ) ]
log Ptrue = 0.16731
Ptrue = 1.47
As for Funnel ( two ) in pH 9,
Concentration of sulfa drug in organic stage = 0.1905 µg mL-1
20 milliliter of organic stage contained a sum of 3.81 µg of sulfa drug
Entire sulfa drug in aqueous stage = 100 µg – 3.81 µg
= 96.19 µg
Papp = ( 3.81/20 ) / ( 96.19/20 )
= 0.039609
log Dat pH 9= log P – log [ 1 + 10 ( pH-pKa ) ]
log Ptrue = log 0.039609 + log [ 1 + 10 ( 9-7.1 ) ]
= 0.50323
Ptrue = 3.1859
Papp = Ptrue ? funionised
0.039609 = 3.1859 ? funionised
funionised = 0.012433
= 1.2433 %
Sulfathiazole is a weak sulfa drug with pKa value of 7.1. Therefore, it is significantly ionised within a physiological pH scope. In this ‘shake-flask ‘ method, the consequence of ionization on the Papp of sulfathiazole can be investigated by mensurating the sum of sulfathiazole being extracted into octanol ( organic stage ) from aqueous stage at different pH. In pH 1, it is evidently noticed that Papp = Ptrue which means that the drug is 100 % nonionized as the funionised = 1. From the computations made from the experiment, the concentration in octanol bed for acidic medium ( pH 1 ) is 2.9796µg mL-1. As for the alkali medium ( pH 9 ) , the concentration in octanol is 0.1905µg mL-1. The concentration difference in different pH is due to ionization of the drug to organize a salt which will finally alter the solubility profile. Papp value in pH 1 is expected to be higher than in pH 9 buffer so it is besides expected that the optical density is higher in acidic medium. There are some human mistakes happening when fixing the criterions. The standard optical density is set to be more than 1.0 which is non ideal for optical density measurings. In order to obtain a more accurate consequence, the optical density should be set less than 1.0.
The advantage of ‘shake-flask ‘ method is that log P can be straight obtained utilizing this method. One of the disadvantages of ‘shake-flask ‘ method is that it is clip devouring to do certain that the aqueous and organic beds are separated to the full. This method requires good research lab accomplishments such as accomplishments in agitating the separating funnels. Separating funnels should be made certain non to be shaken excessively smartly to forestall emulsion from organizing. Laboratory accomplishment such as running off the bottom bed in the funnels is of import to avoid the organic bed from running off together with the aqueous bed.
Determination of Log P values utilizing HPLC
Compound
t0
tr
K = ( tr-t0 ) /t0
Log K
Log P
Sulfacetamide
1.690
3.1
0.8343
-0.07867
-1.0
Sulfadiazine
1.693
4.127
1.4377
0.15766
-0.2586
Sulfamethoxazole
1.693
15.537
8.1772
0.91260
0.9
Sulfapyridine
1.693
5.730
2.3845
0.37740
0.4
Sulfathiazole
1.693
4.840
1.8588
0.26924
0.14554
C26
1.533
127.257
79.3897
1.8998
3.300
Table 5: Log P values obtained utilizing HPLC
Figure 5: Graph of log K versus log P in finding log P values utilizing HPLC.
Based on Figure 5, the equation obtained was y =0.4818x + 0.3096.
As C26 is an unknown compound, the log P is unknown excessively. Therefore, the log P for C26 could be calculated based on the equation obtained.
y=0.4818x + 0.3096
1.8998= 0.4818x + 0.3096
x= 3.300
Log P value for C26 is 3.300
The information follows a consecutive line with R2 of 0.8782. This consecutive line indicates that log K additions with log P. This shows that the keeping clip ( clip taken to elute from HPLC column ) in HPLC is straight affected by the log P of the compound. The higher value of log P causes the longer clip the compound stays in the column due to hydrophobic-hydrophobic interaction.
Sulfadiazine and sulfathiazole have Log P values of 1.3 and -0.4 severally at specific pH of 7.5. This indicates that the log P values are known as Papp which is affected by the ionization to organize a salt. Papp values changed due to the charge as the compound become ionized. The line of a graph would be badly affected with diminishing values of R2 value if Papp values were used to plot the graph. Therefore, Papp should be converted to Ptrue to guarantee the one-dimensionality of the graph ‘s line.
There are jobs in obtaining Log P for unknown sulphonamide utilizing the old HPLC theoretical account system. I could non acquire any consequences even though I have waited for more than two hours. This might due to the ground that C26 is a really hydrophobic compound compared to other sulphonamides. C26 has a Log P of 3.300, therefore it is a really hydrophobic drug. This is proven due to the high keeping clip of 127.257 obtained from the new theoretical account of HPLC system.
Mutual opposition of nomadic stage can impact the separation of the compound as the keeping in reversed-phase liquid chromatography is regulated by interactions in the nomadic stage. Low mutual opposition of nomadic stage will increase the keeping clip of the compound as hydrophobic-hydrophobic interaction in the column longer than expected. In contrast, higher mutual opposition of nomadic stage will diminish the keeping clip of the compound. For case, keeping clip and separation factor will diminish if the concentration of methyl alcohol is increased. This is due to the increased sum of adsorbed organic compound will do weakening of hydrophobic interaction between the solute and adsorbent ( Ching, 2000 ) .
Improvements could be made to the experiment to better the consequences. Perennial measurings such as replicate injections of the same solution can be done to obtain accurate consequences.
The advantages of HPLC method in finding P is that HPLC requires a little sum of sample which does non necessary to hold 100 % pureness. HPLC is more sensitive in observing the signals even in a little alteration of concentration. It is easy to run and is a dependable system. In contrast, it is expensive compared to TLC and ‘shake-flask ‘ method. Merely one sample can be tested at a HPLC system at a clip.
Of all the methods, I think that HPLC method is the best method in finding the log P of sulphonamides. The clip taken to fix the sample for HPLC check is normally short. HPLC can supply a big figure of quantitative and qualitative consequences with a individual analytical tally ( Vogeser et al, 2008 ) .
Determination of chemical pureness
Extremum
Area
1
6.41388
2
13.62792
Table 6: Areas obtained from HPLC for chemical pureness checks
X 100 %
= X 100 %
= 67.9975 %
? 68 %
C26 has a chemical pureness of 68 % . Since C26 is an unknown compound, there is no criterion required. The chemical pureness value of C26 could non be compared with any literature value due to the fact it is an unknown compound.
Decision:
The divider coefficient of sulphonamides can be measured by different methods in chemical science research lab such as TLC, ‘shake-flask ‘ method, and HPLC. From this experiment, it can be concluded that HPLC is the best method in finding the belongingss of sulphonamides due to the fact that it advantages in obtaining accurate consequences.
Mentions
1. C.B. Ching, P. Fu, S.C. Ng, and Y.K. Xu, 2000. Consequence of nomadic stage composing on the separation of Inderal enantiomorphs utilizing a perphenylcarbamate ?-cyclodestrin bonded chiral stationary stage. Journal of Chromatography A. 898. 53-61.
2. Donald Cairns, 2003. Necessities of Pharmaceutical Chemistry. Second Edition. Pharmaceutical Press. Chapter 2. Page 29-36.
2. Krisztina Takacs-Novak, Alex Avdeef, 1996. Interlaboratory survey of log P finding by shake-flask and potentiometric methods. Journal of Pharmaceutical and Biomedical Analysis. 14, 1405-1413.
3. Michael Vogeser and Christoph Seger, 2008. A decennary of HPLC-MS/MS in the everyday research lab – Goals for farther developments. Clinical Biochemistry. 41. 649-662.
4. William O. Foye, Thomas L. Lemke, David A. Williams, 1995. Principles of Medicinal Chemistry. Fourth Edition. Williams & A ; Wilkins. Chapter 34. Page 764.
5. Graham L.Patrick, 2005. An Introduction to Medicinal Chemistry. Third Edition. Oxford. Chapter 16. Page 382-386
