TAKE HOME TEST-CHROMATOGRAPHY GRADUATE PROGRAM OF PHARMACY, UNIVERSITY OF PANCASILA, JAKARTA 1. In an aphrodisiac drink, the powder formula consists of coffee, and Pasak Bumi roots. However, the National Agency for Drug and Food Control suspected that this formula contains Sidenafil and Tadalafil. Design an experiment using various chromatographic methods that this formula is containing coffee, Pasak Bumi, and contaminated by Sidenafil and Tadalafil. 2. There is an MLM honey product called Propulis. This Propulis contains flavonoids, and aminoacids. Using various chromatographic method, you have to design an experiments, what are the amino acids, and the flavonoids. Predict the bees have taken the honey from flowers of which plants. 3. It was known that a various pheophorbides derived chlorophyll were active for anti hepatitis virus. Design an experiment using various chromatographic methods, determining the pheophorbides. 4. An expensive perfume was made of cananga, eagle wood and vetiver oil. Design an experiment using chromatographic methods that this perfume containing these essential oils. Determine how to quantify the major compound of each essential oil. 5. Various fruits are claimed containing rich of antioxidant, phenol, flavonoid, and tannin. Design a combined chromatographic and spectroscopy experiments to
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
TAKE HOME TEST-CHROMATOGRAPHY
GRADUATE PROGRAM OF PHARMACY, UNIVERSITY OF PANCASILA,
JAKARTA
1. In an aphrodisiac drink, the powder formula consists of coffee, and Pasak Bumi roots.
However, the National Agency for Drug and Food Control suspected that this formula
contains Sidenafil and Tadalafil. Design an experiment using various
chromatographic methods that this formula is containing coffee, Pasak Bumi, and
contaminated by Sidenafil and Tadalafil.
2. There is an MLM honey product called Propulis. This Propulis contains flavonoids,
and aminoacids. Using various chromatographic method, you have to design an
experiments, what are the amino acids, and the flavonoids. Predict the bees have taken
the honey from flowers of which plants.
3. It was known that a various pheophorbides derived chlorophyll were active for anti
hepatitis virus. Design an experiment using various chromatographic methods,
determining the pheophorbides.
4. An expensive perfume was made of cananga, eagle wood and vetiver oil. Design an
experiment using chromatographic methods that this perfume containing these
essential oils. Determine how to quantify the major compound of each essential oil.
5. Various fruits are claimed containing rich of antioxidant, phenol, flavonoid, and
tannin. Design a combined chromatographic and spectroscopy experiments to
evaluate these claims, antioxidant capacity, total phenolic, flavonoids and tannin
content. Design equivalency for each constituent, namely, of antioxidant with
Ascorbic Acid, phenol with galic acid, flavonoid with quercetin and tannin with
cathecin.
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
ANSWER SHEETS
1. In an aphrodisiac drink, the powder formula consists of coffee, and Pasak Bumi roots.
However, the National Agency for Drug and Food Control suspected that this formula
contains Sidenafil and Tadalafil. Design an experiment using various
chromatographic methods that this formula is containing coffee, Pasak Bumi, and
contaminated by Sidenafil and Tadalafil.
Answer :1
Analysis of sildenafil and tadanafil can use the method of HPLC & LC- MS with a
column C18. While the tools and materials used are: Sildenafil citrate and tadalafil
use as standar, Methanol, acetonitrile (analytical grade) and formic acid. Method &
procedure is :
a. Recovery experiments were performed to evaluate the reliability and suitability of
the optimized conditions coffee. In this study, blank coffee powder was spiked
with 10 μg/g standard concentration. Two different types of extraction solvents
which were acetonitrile (A) and methanol (M) with two different extraction steps;
without nitrogen evaporation and with nitrogen evaporation step as used were
evaluated.
b. Extraction with evavoration : A 1 g sample was weighed into 50 mLcentrifuge
tube, added with 10 mLsolvent and being shake by overhead, sonicated and
centrifuged at 4000 rpm. The supernatant was then filtered with 0.2 μm PVDF
micro filter and injected into instruments. Result collected from this method were
labelled as A1 (using acetonitrile as extraction solvent) and M1 (using methanol
as extraction solvent).
c. Extraction with evaporation step : The first part of the extraction were the same as
above mentioned, however following the centrifugation step, the sample
supernatant collected was further evaporated using nitrogen evaporator until
complete dryness, reconstituted with 1 mL acetonitrile: water (1:1), filtered and
ready for instrumentation. Result collected from this method were labelled as A2
(using acetonitrile as extraction solvent) and M2 (using methanol as extraction
solvent).
1
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
d. Optimization of sample extraction : Two different extraction techniques; without
the evaporation step and with evaporation step were applied in order to choose the
most efficient technique to analyze coffee sample. On both methods, two type of
extraction solvent were tested, acetonitrile (A) and methanol (M).. However,
coffee contains more matrix interferences such as fat, caffeine, sugar and we
presumed much lower concentration of adulterant added (if any) as compared to
pharmaceutical and herbal products. Thus, the methodology was optimised in
order to improve the efficiency for our samples. Modification aimed to obtain
more concentrated extract, thus the amount of sample weighed were increased to
one gram and the final extract was not diluted as recommended. The only
modification made on this method was the replacement of dichloromethane:
isopropanol (9:1) with either acetonitrile or methanol as extraction solvent, since
most studies reported the efficiency of these solvents in the analysis. The mean
recoveries and standard deviation for all four methods attempted is illustrated in
Tables 1 and 2. All analytes by HPLC-DAD displayed recovery of more than 80%
and 30% to 102% from LC-MS-TOF. The standard deviation was less than 10%
for all samples extracted and detected by LC-MS-TOF, but only sample extracted
with acetonitrile (Method A1 and A2) demonstrated standard deviation of less
than 10% by HPLC-DAD. Methanol was found not suitable to extract PDE5
inhibitors in coffee, as it generates very high interferences with DAD detection.
That acetonitrile proved a good recovery results as compared with the methanol
which generated higher background noise. The report also stated that acetonitrile
was necessary solvent for tadalafil due to its poor solubility in methanol.
e. Optimization of HPLC : attempted using two different combinations of mobile
phase: The combination of water and acetonitrile with modification on gradient
elution programme to obtain the best separation and the combination of water and
methanol with modification on gradient elution programme to obtain the best
separation. The selected combination of mobile phase was further optimized by
two different matrix modifier to find the most suitable matrix modifier in this
analysis: 10 mM ammonium format and 0.1% formic acid. Chromatogram data
were collected from 190 nm to 500 nm wavelength, within 20 min of run time.
The UV signal were monitored at 230 nm, 245 nm and 290 nm.
2
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
f. Selection of mobile phase for HPLC analysis :The combination of water and
methanol as the mobile phase are able to separate well tadalafil from vardenafil
and sildenafil with high intensities. However, the background noise is noticibly
high and affect the identification of the analytes of interest. The peak of vardenafil
and sildenafil however are not separated well with the use of methanol as both
analytes are very similar in the molecular structure and its polarity. The peak of
vardenafil and sildenafil are not well separated when methanol is used as mobile
phase. The use of acetonitrile and water however provide better separation with
high intensities for all three compounds. All three peaks of vardenafil, sildenafil
and tadalafil were distinguishly separated. The peak of caffein is also separated
from the three interested compounds. At the same time, the background noise is
not too significant as to interfere with the peak of interest. The presence of several
Natoms in the molecular structure of target compounds would cause serious peak
tailing if no modifier was added into mobile phase in HPLC analysis. Ammonium
formate was used as matrix modifier to separate sildenafil, tadalafil and other
drugs in herbal products by LC-MS/MS. It was found in this study that the use of
ammonium format was not suitable to separate both sildenafil and vardenafil by
HPLC. Peaks of vardenafil and sildenafil found to be overlapping with each other
and these two compounds failed to be separated either by isocratic or gradient
elution. The use of ammonium formate was presumably suitable to analyze both
sildenafil and vardenfil simultaneously if using mass spectrometry because the
chromatogram of each target compound can be extracted individually, but not
with DAD detector. The separation problem was resolved by adding formic acid
in mobile phase is recommended. It was found that the use of 0.1% formic acid
was able to separate these two peaks of sildenafil and vardenfil. It was reported
that mobile phase in acidic condition help to improve the sensitivity of sildenafil
in liquid chromatography. As a conclusion, the use of acetonitrile and water with
0.1% formic acid as matrix modifier added to both solution was found to be the
most suitable mobile phase in this study.
g. Optimization of gradient elution: A linear solvent gradient using 0.1% formic acid
in water and 0.1% formic acid in acetonitrile with flow rate of 1 ml/min was the
most suitable combinations in terms of analysis time, resolution and sensitivity.
3
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
Best separation obtained when composition of 0.1% formic acid in acetonitrile
was linearly ramped from 3% to 80% in 10 min and being maintained within 5
min before dropped instantly to 3% for another 5 min. Prominent peaks of
vardenafil, sildenafi l and tadalafi l were resolved at 8.459 min, 9.109 min and
11.157 min, respectively. The compounds were identified by comparing the
retention time with the individual compounds retention time.
h. Wavelength selection : The wavelengths of 290 nm and 230 nm were commonly
selected to quantitate. It was found that at 290 nm the peak intensities of all
analytes were highest as compared with 230 nm and 245 nm. However in coffee
sample, interferences signal was also higher and made quantification more
difficult as it is very close to the compound of interest. The intensities of
background matrix interference were less at 230 nm and yet peak intensities of
analytes of interest were very small. Thus, the identification and quantitation of
vardenafil, sildenafil and tadalafil in coffee was selected at 245 nm as it provided
prominent peak intensities with least matrix interferences and more stable
baseline.
i. Optimization of LC-MS-TOF : The developed HPLC method was then transferred
to LC-MS-TOF analysis but the gradient programme was optimized to get best
separation. The mass spectrometer was operated in the positive ion mode.
Nitrogen gas was utilized as the nebulizer gas and electrospray ionization (ESI)-
low concentration tuning mix solution used as the calibrations solution. All the
masses were corrected using internal reference ions of m/z 322.0481, 622.0289
and 922.0098 with mass error less than 5 ppm.
j. Statistical analysis : The results were expressed as mean value ± standard
deviation of three replicates. Analysis of sample was performed using SPSS
software version 11.5 2002 to determine the variation of recovery among four test
methods and individual analyte. The level of significance was p<0.05.
k. Evaluation of method performance : Statistical analysis showed that all four
extraction methods were significantly different (p<0.05) for both instruments,
HPLC-DAD and LC-MS-TOF. Further statistical analysis found that tadalafil was
significantly different (p<0.05) for all four methods detected with HPLC-DAD as
can be seen in Table 1. However, sildenafil and vardenafil were not differently
4
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
significant (p>0.05) by method A1 and A2. Result of LC-MS-TOF showed that
tadalafil and sildenafil were not significantly different (p>0.05) for method M1
and A2, yet differ significantly (p<0.05) for method A1 and M2 as indicated in
Table 2. Vardenafil found to be significantly different (p<0.05) for all four
methods. According to the guideline on the acceptance of method performance by
European Union (2002/657/EC), the accuracy must be within 80% and 110% for
sample spiked at concentration of 10 μg/g. Therefore, methods A1 and M1
fulfilled the criteria for acceptance of performance using LC- MS-TOF whereas
method A1 and A2 by HPLC-DAD. Methods A1 and A2 were finally selected for
additional statistic analysis due to quantitation to be conducted by HPLC-DAD.
Based on statistical test between obtained result and target value (100%), it was
proven that there was no significant difference (p>0.05) for all analytes extracted
by method A1 by HPLC-DAD. However, method A2 showed there were no
significant different (p>0.05) only for vardenafil, whereas sildenafil and tadalafil
were significantly different (p< 0.05). Based on the presented results, method A1
was finally selected for future studies to determine the presence of tadalafil,
sildenafil and vardenafil simultaneously in coffee. Furthermore, the extraction
technique was faster as compared with method A2, which took 3 h longer. Figure
1(b) illustrates a HPLC chromatogram of spiked sample at concentration of 0.5
mg/kg which was extracted using method A1 and Figure 2(b) for LC-MS-TOF
extracted ion chromatogram.
5
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
The total antioxidant capacity of the fractions was determined by phosphomolybdate
method using ascorbic acid as a standard. An aliquot of 0.1 ml of sample solution was
mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate
and 4 mM ammonium molybdate). The tubes were capped and incubated in a water
bath at 95°C for 90 min. After the samples had cooled to room temperature, the
absorbance of the mixture was measured at 765 nm against a blank. A typical blank
contained 1 ml of the reagent solution and the appropriate volume of the solvent and
incubated under the same conditions. Ascorbic acid was used as standard.
b. Total phenolic content of fruits
The total phenolic contents of fruits were estimated using the Folin–Ciocalteu
method, which relies on the transfer of electrons from phenolic compounds to the
Folin–Ciocalteu reagent in alkaline medium, and is a simple, rapid and reproducible
method. In addition, it should be pointed out that some non-phenolic reducing
compounds, such as organic acids and sugars, could interfere the determination of
total phenolic contents by the Folin-Ciocalteu method, which leads to an
overvaluation of the phenolic content. Furthermore, different phenolics might present
different responses with the Folin Ciocalteu reagent, such as gallic acid and rutin have
18
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
similar behaviours, but several flavonoids present low absorption, which leads to an
underestimation of various compounds.
Estimation of total phenolic content
The total phenolic content was determined by the spectrophotometric method. In
brief, a 1 ml of sample (1 mg/ml) was mixed with 1 ml of Folin-Ciocalteu’s phenol
reagent. After 5 min, 10 ml of a 7% Na2CO3 solution was added to the mixture
followed by the addition of 13 ml of deionized distilled water and mixed thoroughly.
The mixture was kept in the dark for 90 min at 23°C, after which the absorbance was
read at 750 nm. The TPC was determined from extrapolation of calibration curve
which was made by preparing gallic acid solution. The estimation of the phenolic
compounds was carried out in triplicate. The TPC was expressed as milligrams of
gallic acid equivalents (GAE) per g of dried sample.
c. Total Flavonoid
Total flavonoid content was determined in quercetine. In a 10 ml test tube, 0.3 ml of
extracts quercetine, 3.4 ml of 30% methanol, 0.15 ml of NaNO2 (0.5 M) and 0.15 ml
of AlCl3.6H2O (0.3 M) were mixed. After 5 min, 1 ml of NaOH (1 M) was added.
The solution was mixed well and the absorbance was measured against the reagent
blank at 506 nm. The standard curve for total flavonoids was made using rutin
standard solution (0 to 100 mg/l) under the same procedure as earlier described. The
total flavonoids were expressed as milligrams of rutin equivalents per g of dried
fraction.
d. Test for tannins
i. 50 mg of catechine was boiled in 20 ml of distilled water and filtered. A few
drops of 0.1% FeCl3 was added in filtrate and observed for colour change;
brownish green or a blue-black colouration was taken as evidence for the
presence of tannins.
ii. The tannins were determined by Folin and Ciocalteu method. 0.1 ml of the
sample extract was added with 7.5 ml of distilled water and adds 0.5 ml of
Folin Phenol reagent, 1 ml of 35% sodium carbonate solution and dilute to 10
ml with distilled water. The mixture was shaken well, kept at room
temperature for 30 min and absorbance was measured at 725 nm. Blank was
prepared with water instead of the sample. A set of standard solutions of gallic
19
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
acid is treated in the same manner as described earlier and read against a
blank. The results of tannins are expressed in terms of gallic acid mg/g of
extract
20
Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)
REFERENCES
1. A.M. Zailina,AM, Aminah, A. & Ambar, YM, Optimization of Extraction Methods for Determination of Phosphodiesterase-5 (PDE5) Inhibitors in Premix Coffee. Sains Malaysiana 42(2)(2013): 135–142.
2. Coneac, G, et al. Flavonoid Contents of Propolis from the West Side of Romania and Correlation with the Antioxidant Activity. Chem. Bull. "POLITEHNICA" Univ. (Timisoara). Volume 53(67), 1-2, 2008.
3. Yoshitake, Y, et al.Determination of Pheophorbide a and Methylpheophorbide a by Fuorosence –HPLC : Availability of Narrow Baseline Method. Analyitical Science Vol 2. 1986.
4. Mariot, PJ, et al. Gas chromatographic technologies for the analysis of essential oils. Journal of Chromatography A, 936 (2001) 1–22.
5. Sacchetti, Gianni et al,. Comparative Evaluation of 11 Essential Oils of Different Origin as Functional Antioxidants, Antiradicals, and Antimicrobials in Foods.Universita Degli Studi di Ferrara. Italy. 2004.
6. Li Fu et al. Antioxidant Capacities and Total Phenolic Content of 56 Wild Fruits from South China. Sun Yat-Sen University. Guangzhou, China. 2010 (Artikel dari www.mdpi.com/journal/molecules.com diakses tanggal 2 Januari 2014.)
7. Sae et al. Antioxidant activity, Total Phenolic, and Total FlVONOID Contents of Whole Plant Extracts Torilis leptophylla L.BMC Complementary and Alternative Medicine. 2012., 12:221 (http://www.biomedcentral.com/1472-6882/12/221 diakses tanggal 2 Januari 2014)