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Theobromine is the primary alkaloid present in the cocoa and chocolate. Theobromine is found in the shells and beans of the cacao plant and it is extracted from the husks of the bean and used for the synthesis of caffeine. The plant species that contain large amount of theobromine are
§ Theobroma cacao
§ Theobroma bicolor
§ Ilex paraguariensis
§ Camellia sinensis
§ Cola acuminate
§ Theobroma angustifolium
§ Coffea Arabica
3, 7-dimethylxanthine, 3, 7-dihydro-3, 7-dimethyl-1H-purine-2, 6-dione
Chemical Formula: C7H8N4O2
Molecular mass: 180.1658 grams
Density: each cacao seed is 2% theobromine
Solubility: 1 gram is soluble in 2.2L of 95% ethanol. It is insoluble in benzene, ether, chloroform and Carbon tetra chloride.
Water solubility: 1 gram is soluble in approximately 2 Lt of water 150 ml of boiling water.
Natural state: solid, a white powder
Melting point: 290-2950C
Boiling point: -2950C
Metabolism: Theobromine is metabolised into methylxanthine and subsequently into methyl uric Acid .The main important enzymes include CYP1A2 and CYP2E1. 
Half life: 7.1±0.7 hours
Cocoa powder such as Hershey's cocoa contains 108mg(2.16%) of Theobromine per tablespoon (5gram) of powder. Chocolates contain 0.5-2.7% of Theobromine.theobromine can also found small amounts in the kola nut(1.0-2.5%),the guarana berry and the tea plant. White chocolates contain trace amount of Theobromine.
The Russian chemist Alexander Woskresensky first discovered Theobromine in 1841 in cacao beans. Hermann Emil Fischer synthesized Theobromine from xanthine. After following study in the late 19th century it was put to use in 1916, where it is recommended by the publication principles of medical treatment.
Theobromine is derived from the name theobroma ,which itself made up of Greek words theo(god) and brosi(food),meaning “food of the gods” .with the suffix ine given to the alkaloids and other basic nitrogen-containing compounds.
Mechanism of action:
Theobromine is a potent cyclic adenosine monophospate (cAMP) phosphodiesterase inhibitor. It inhibits the enzyme phosphodiesterase that converting active cAMP to inactive form, which results in increased cAMP and release of catecholamines.When theobromine, inhibits the inactivation of cAMP,the effects of neurotransmitter which stimulated the production of cAMP were long lived.so,the net result is stimulatory effect.cAMP works as a secondary messenger in many hormone and neurotransmitter controlled systems such as break down of glycogen.
Theobromine is also used in cough medicine as a potential antitussive. It also inhibits capsaicin induced sensory nerve depolarization of guinea pig vagus and human vagus nerve in vitro. And in recent study indicate that it acts on the vagus nerve, which runs from the lungs to the brain.
The chocolates contains small amount of theobromine,that chocolates can safely consumed by humans in large quantities. But animals slowly metabolised theobromine can easily consumed in small quantities can cause poisoning. The most victims for poisoning are dogs. Cats and kittens are more sensitive and cats are less prone to eat chocolate because they are unable to taste sweetness.
Theobromine is especially toxic to cats, horses, parrots, voles, dogs and cats because they are unable to metabolised chemical effectively. If these animals take the chocolates they will remain in blood stream for 20 hours. Medical treatment is performed to induce vomiting after 2 hours by administering and injecting barbiturates s and benzodiazepines for seizures, antiarrhythmics for heart arrhythmia sand fluid diuresis.
The clinical signs for theobromine poisoning are nausea, vomiting,diarrhea and increased urination and increased blood pressure . Sometimes bradycardia or most commonly tachycardia occurs and myocardial dysrhythmias, especially ventricular premature beats.Central nervous system excitability, excitement, tremors, seizures and ultimately coma, Death occurs within 6-24 hours with acute exposure.
HPLC (High pressure liquid chromatography):
HPLC is the most common technique used in analytical and biochemistry to separate and quantification of drugs in pharmaceutical formulations.
The basic principle involved in the HPLC is to pass the mixture of analyte through the stationary phase contained column by pumping the mobile phase at high pressure through the column and the detector detects retention time.
The time taken to elute analyte from the column is retention time. It is a consider as a characteristic of a molecule.
The time between sample injection and an analyte peak reaching a detector at the end of the column is termed theretention time(tR) . It is a characteristic of the solute or molecule.
Reverse phase HPLC :
Reverse phase HPLC is used in pharmaceutical industries to qualify the drugs before their release.
Reverse phase HPLC consists of a polar mobile phase and non-polar stationary phase. The silica which is present in the stationary phase modify it to non-polar by attaching with the long hydrocarbon chains contain either 8 or 18 carbon atoms.
Reverse phase chromatography works on the principle of hydrophobic forces between the solute molecules of mobile phase and immobilised hydrophobic ligand..in that non-polar molecules tend to form attractions with hydrocarbon groups because of vander wall forces.
Non-polar compounds are less soluble in solvents because they need to break the hydrogen bonds because they squeeze in the water and methanol. So, the non-polar compounds have longer retention time and polar compounds have smaller retention time.
1. Quantification of drugs and their metabolites in biological fluids.
2. Determination of partition coefficients and pKa of drugs and of drug protein binding.
3. The combination of high-pressure liquid chromatography (HPLC) with UV/visible detection provide a accurate, precise and robust method for quantitative analysis of pharmaceutical products in the industry.
4. Monitoring of the stability of pure drug substances and of drugs in formulations, with quantification of any degradation products.
Mass spectroscopy is an analytical technique used for separating and identifying the molecular mass of the compound by their mass to charge (m/z) ratio.
This modern analytical chemistry technique is the combination of physical separation capability of high performance liquid chromatography (HPLC) and the mass spectrometry capability of analysing the mass of the compound of interest. This technique is vastly used in drug development studies like quantitative bioanalysis, quality control, impurity identification, glycoprotein and peptide mapping, metabolite identification, In vivo drug screening etc., because of its very high sensitivity and selectivity.
The main advantage of mass spectroscopy is to identify the unknown compounds by using picogram (10-12) amounts.
Mass spectrometer contains three fundamental components namely ion souce, mass analyser and detector.
* Mass spectroscopy provides a highly specific method for determining or confirming the identity or structure of drugs and raw materials used in their manufacture.
* Mass spectroscopy has become a important tool in proteomics, which is currently a major tool in drug discovery.
* Mass spectroscopy in conjunction with gas chromatography (GC-MS) or liquid chromatography (LC-MS) provides a method for characterising the impurities in drugs and formulation excipients.
* GC-MS and LC-MS provide a highly specific and sensitive method for determining the drugs and their metabolites in biological fluids and tissues.
SOLID PHASE EXCTRACTION:
Solid phase extraction is an extraction method that uses a solid phase and liquid phase to isolate a analyte from the solution. SPE is usually used to clean up the sample before using a chromatographic method to quantify the amount analyte in the sample.
Solid phase extraction is useful for selective separation of interferants from analytes, which is not achievable by liquid/liquid extraction.
Solid phase extraction is widely used in bio analytical measurements and environmental monitoring for concentrating trace amounts of analytes.
In clinical applications, SPE is used to monitor the drug residues in urine, blood and other body fluids.
SPE is used to monitor traces of accelerates in fire debris in forensic department.
Small volumes of solvents required for both washing and elution .
Emulsions will not form between two phases like liquid/liquid extraction.
Extractions can be carried out in batches rather than serially.
Reverse phase SPE:
Reverse phase SPE separate the analytes based on their polarity. Reverse phase SPE involves moderately polar or polar mobile phase and non-polar stationary phase.the catrigade contains Due to the hydrophobic effect the low to mild polar compounds retain on the SPE cartiage.
The term validation is defined as the process of establishing
“The process of establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting pre-determined specifications and quality characteristics”.
Alternatively, validation can be considered as the documented activity performed to demonstrate that a given utility, system, process or piece of equipment does what it purports to do.
Thus validation can be applied to many different stages of manufacture, e.g. process validation, cleaning validation, analytical validation.
Generally validation is applied to different types of analytical test methods
i. Quality control tests( for finished product)
ii. In process control methods(for intermediate, bulk product)
iii. Acceptance of test methods(for raw material)
iv. Methods used for measuring the pharmaceutical substances in biological samples
v. Methods used in research and development for new medicine.
To ensure that all pharmaceutical manufacturers validate their analytical methods to the highest standards, a set of standards has been developed and agreed internationally for use in the pharmaceutical industry. These have been produced by the International Conference on Harmonisation (ICH) which has produced guidelines in a number of aspects of pharmaceutical quality.
ICH Q2 (R1) is the revised standard for “Validation of Analytical Procedures: Text and Methodology”. According to ICH Q2(R1), analytical validation should be carried out by assessing the following criteria (criterion singular) of the method
o Intermediate precision
o Limit of detection(LOD)
o Limit of quantification(LOQ)
* Stability of analytical solution
The time to carry out the full validation is about 4-6 weeks for the analyst.
The accuracy of analytical procedure expresses the closeness agreement between the value that is accepted either as a conventional true value or accepted reference value. 
The ICH recommends that a minimum of nine determinations over a minimum of three concentrations are used to demonstrate the accuracy.
Accuracy can be expressed in terms of percentage recovery, where
% Accuracy = Mean observed concentration x 100
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions
It is generally considered at three levels. They are reproducibility, intrinsic precision and repeatability.
The precision of an analytical method is usually expressed as the Standard Deviation, Relative Standard Deviation (RSD) or Coefficient of Variation (CV) of the method,
RSD (CV) = Standard Deviation x 100 %
Recall from statistics that 68% of results in a series of measurements lie within the range
Mean ± 1 standard deviation
And that approx 95% of results lie within
Mean ± 2 standard deviations
It expresses the precision under same operating conditions in a short interval time to get the same results for the same batch. it also known as intra-assay precision. The repeatability of analytical methods used in the QC of drugs and medicines should be high.
At least 5 or 6 determinations of three matrices at two or three concentrations should be done.
It expresses the precision of the analytical method obtained within a single laboratory by using different operators and testing the same batch of materials at different times using different equipments and different reagents. It is calculated as RSD.
It expresses the precision between the laboratories. There are variations like difference in humidity and temperature, variation in material and conditions
“The Linearity of an analytical procedure is its ability to obtain test results that are directly proportional to the concentration of analyte in the sample within a given range”.
The linearity should be measured over 0-150% of the expected concentration of the drug. The linearity is determined by a series of 3 to 6 injections of 5 or more standards.
The advantage of having a proportional relationship is that only a single standard solution is needed to construct the calibration graph--the origin sets the second data point. If a linear but non-proportional graph is obtained at least two and preferably five standard solutions are needed for the calibration graph.
For those calibration curves that are not straight lines (rectilinear), another mathematical relationship may be used but the relationship must be demonstrated (e.g. a quadratic or curvilinear relationship).
The ICH recommends for accuracy reporting the calibration curve must contain correlation co-efficient,y-intercept, slope of the regression line and residual sum of squares.
“The range of an analytical procedure is the interval between the upper and lower concentrations (or amounts) of analyte in the sample for which it has been demonstrated that the method has an acceptable degree of accuracy, precision and linearity
For assay tests ICH requires the minimum specified range of to be 80 to 120 percent of the test concentration.
“Robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.”
Ideally, a method should not be sensitive to small variations in temperature, pH, the concentrations or purities of the reagents etc. or the time of reaction (if a reaction is necessary)
Robustness can be measured by making small changes in the parameters of the analytical method, like pH, flow rate, mobile phase composition, temperature, injection volume, wavelength.
Sensitivity is the name given to that attribute of an analytical method or procedure that indicates how little of the analyte can be measured with the desired accuracy and precision.
It is important that the routine analytical method should be sufficiently sensitive for measuring the analyte in the sample, e.g. intermediate formulations, and bulk and finished products, so that the method will be able to detect, quantitatively, any concentrations that substantially deviate from the specification.
ICH Q2(R1) requires sensitivity to be evaluated in two ways
4.1 Limit of Detection
“The detection limit (limit of detection, LoD) is the lowest amount of the analyte in a sample that can be detected , but not necessarily quantified as an exact value” (ICHQ2(R1)
Limit of Quantitation (LoQ)
“The quantitation limit of an individual analytical procedure is the lowest amount of the analyte in a sample that can be quantitatively determined with suitable precision and accuracy”. The LoQ is higher than LoD.
Specificity is the ability to measure the analyte qualitatively and/or quantitatively without interference from any other substance. A method that lacks specificity will suffer interference from other components in the sample and give results that inaccurate.
System suitability tests:
System suitability tests is used to verify the resolution,column effiency and repeatability of a chromatographic system to ensure its adequacy for particular method[.6]
System suitability tests involves the comparsion of chromatographic trace with a standard trace under ideal conditions. These allows the comparison of peak shape, peak width and baseline resolution.
The primary SST parameters are resolution,repeatability,column efficiency and tailing factor. These are most important to indicate as they indicate system stability, precision and system specificity.
System suitability test contains the tests like
Ø Number of theoretical plates(efficiency)
Ø Relative standard deviation(precision)
Ø Capacity factor
Ø Separation(relative retention)
Ø Tailing factor
Plate number or number of theoretical plates:
Plate number is the measure of the sharpness of the peaks and efficiency of the column. Number theoretical plates measured in various ways by using peak width at the base and peak width at half of the peak height.
N = no. of theoretical plate
RT = retention time
W = width of peak
w1/2is the peak width at half height.
If the length of the column is L,then HETP is
Where N=number of theoretical plates and
L=length of the column
The higher the N and smaller the HETP gives better efficiency.
Capacity facror used to describe the migration rate of an analyte on column.it is also called as retention factor.
Capacity factor value gives the indication of how long the each component wae retained on column
Capacity factor can be calculatd by using the formula
Where Tr=retention time of the peak of intrest
T0=retention time of the unretained peak
When the analyte capacity factor is less than one ,elution is so fast that accurate determination of retention time is difficult and retention factor more than 20 menas elution takes long time.
Ideally capacity factor is in between 1 to 5.
Separation factor describes the separation of two peaks on column. It is calculated by using capacity factor because peak separation is depends upon the components interaction with stationary phase.
If we consider the A and B as two peaks,then
Separation factor(α) =KB/KA
Where,KA =capacity factor for A
KB=capacity factor for A
When A elutes faster than B. the separation factor is always greater than one.
Peak resolution is used in separation between peak and efficiency of column. Peak resolution is express ratio of distance between the two peaks to the mean value of width of peak.
And peak resolution is calculated by a following equation.
RS = 2(TR2 – TR1/ W2 + W1)
Where, TR2 = retention time of your late eluting peak
TR1 = retention time of your early eluting peak
W2 = width of your late eluting peak
W1 = width of your early eluting peak
Base line resolution is achieved when R=1.5
This is a measure for the asymmetry of the peak.
It is defined as the distance from the front slope of the peak to the back slope divided by twice the distance from the center line of the peak to the front slope, with all measurements made at 5% of the maximum peak height..
RELATIVE STANDARD DEVIATION:
Preparation of sample:
Preparation of mobile phase:
To a conical flask add 820 ml of water and 170 ml of methanol ,
RESULTS AND DISCUSSION:
1st run – 298.133
2nd run – 237.803
3rd run – 297.335
1st run – 497.8
2nd run – 497.472
3rd run - 497.871
1st run – 715.5
2nd run – 714.536
3rd run – 785.163
1st run – 969.99
2nd run – 969.293
3rd run – 969.385
1st run – 1223.124
2nd run – 1223.599
3rd run – 1222.733
1st run – 1473.126
2nd run – 1472.75
3rd run – 1475.189
1strun – 1800.511
3rdrun – 1802.75
Residual analysis statical out put of standard Theobromine from HPLC-UV:
Standard theobromine regression analysis from HPLC-UV is given below.
Adjusted R Square
Residual analysis of standard Theobromine:
The calibration curve calculated by regression analysis using the equation
Where Y= response of midpoint selected on the calibration curve,
m = mean slope coefficient,
x = the concentration for the calibration line equation,
c = y- intercept for mean slop coefficient
by placing the values in above equation,
. Gurudeep Chatwal, Sham K.Anand (2007). Instrumental Methods of Chemical Analysis. 5th ed. India: Himalaya Publishing House. p 2.270-2.302
Solid phase extraction: http://www.springerlink.com/content/h72xx3624q122085/fulltext.pdf http://www.chemistry.adelaide.edu.au/external/soc-rel/content/spe.htm
Validation: 6) Michael Dong, Roy Paul, and Lea Gershanov. (sepetember 2001). Getting the peaks perfect: System suitability for HPLC On-line testing can ensure data quality in pharmaceutical assays..todays's chemistry at work. 10 (01), 38-40,42.
8. http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm http://www.microsolvtech.com/calculators_chrom.asp#
R.A. van Iterson Drenthe College Emmen Holland for www.standardbase.com
A Guide to Validation in HPLC
Based on the work of G. M. Hearn PERKIN ELMER
An introduction to analytical method validation
By - 05/20/2007
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