Theobromine

Beginnings:

Theobromine is the primary alkaloid nowadays in the chocolate and cocoa. Theobromine is found in the shells and beans of the chocolate tree works and it is extracted from the chaffs of the bean and used for the synthesis of caffeine. The works species that contain big sum of theobromine are

§ Theobroma chocolate tree

§ Theobroma bicolor

§ Ilex paraguariensis

§ Camellia sinensis

§ Cola acuminate

§ Theobroma angustifolium

§ Guarana

§ Coffea Arabica

Synonym:

3, 7-dimethylxanthine, 3, 7-dihydro-3, 7-dimethyl-1H-purine-2, 6-dione

Xantheose

Diurobromine

Chemical Formula: C7H8N4O2

Molecular mass: 180.1658 gms

Density: each chocolate tree seed is 2 % theobromine

Solubility: 1 gm is soluble in 2.2L of 95 % ethyl alcohol. It is indissoluble in benzine, ether, trichloromethane and Carbon tetra chloride.

Water solubility: 1 gm is soluble in about 2 Lt of H2O 150 milliliter of boiling H2O.

Natural province: solid, a white pulverization

Melting point: 290-2950C

Boiling point: -2950C

Metamorphosis: Theobromine is metabolised into methylxanthine and later into methyl uric Acid.The chief of import enzymes include CYP1A2 and CYP2E1. [ 1 ]

Half life: 7.1±0.7 hours

Elimination: renal

Measure:

Cocoa pulverization such as Hershey ‘s chocolate contains 108mg ( 2.16 % ) of Theobromine per tablespoon ( 5gram ) of pulverization. Chocolates contain 0.5-2.7 % of Theobromine.theobromine can besides establish little sums in the goora nut nut ( 1.0-2.5 % ) , the guarana berry and the tea works. White chocolates contain trace sum of Theobromine.

History:

The Russian chemist Alexander Woskresensky foremost discovered Theobromine in 1841 in cacao beans. Hermann Emil Fischer synthesized Theobromine from xanthine. After following survey in the late nineteenth century it was put to utilize in 1916, where it is recommended by the publication rules of medical intervention.

Theobromine is derived from the name Theobroma, which itself made up of Grecian words theo ( God ) and brosi ( nutrient ) , intending “food of the gods” .with the suffix ine given to the alkaloids and other basic nitrogen-containing compounds.

Mechanism of action:

Theobromine is a powerful cyclic adenosine monophospate ( camp ) phosphodiesterase inhibitor. It inhibits the enzyme phosphodiesterase that change overing active camp to inactive signifier, which consequences in increased camp and release of catecholamines.When theobromine, inhibits the inactivation of camp, the effects of neurotransmitter which stimulated the production of camps were long lived.so, the net consequence is stimulatory effect.cAMP works as a secondary courier in many endocrine and neurotransmitter controlled systems such as break down of animal starch. [ 2 ]

Theobromine is besides used in cough medical specialty as a possible antitussive. It besides inhibits capsaicin induced centripetal nervus depolarisation of guinea hog pneumogastric and human pneumogastric nervus in vitro. And in recent survey indicate that it acts on the pneumogastric nervus, which runs from the lungs to the encephalon.

Theobromine toxic condition:

The cocoas contains little sum of theobromine, that cocoas can safely consumed by worlds in big measures. But animate beings easy metabolised theobromine can easy consumed in little measures can do toxic condition. The most victims for poisoning are Canis familiariss. Cats and kitties are more sensitive and cats are less prone to eat cocoa because they are unable to savor sugariness.

Theobromine is particularly toxic to cats, Equus caballuss, parrots, field mouses, Canis familiariss and cats because they are unable to metabolised chemical efficaciously. If these animate beings take the cocoas they will stay in blood watercourse for 20 hours. Medical intervention is performed to bring on purging after 2 hours by administrating and shooting barbiturates s and benzodiazepines for ictuss, antiarrhythmics for bosom arrhythmia sand fluid diuresis.

The clinical marks for theobromine toxic condition are sickness, purging, diarrhoea and increased micturition and increased blood force per unit area. Sometimes bradycardia or most commonly tachycardia occurs and myocardial dysrhythmias, particularly ventricular premature beats.Central nervous system irritability, exhilaration, shudders, ictuss and finally coma, Death occurs within 6-24 hours with acute exposure.

HPLC ( High force per unit area liquid chromatography ) :

HPLC is the most common technique used in analytical and biochemistry to divide and quantification of drugs in pharmaceutical preparations.

Principle:

The basic rule involved in the HPLC is to go through the mixture of analyte through the stationary stage contained column by pumping the nomadic stage at high force per unit area through the column and the sensor detects keeping clip.

The clip taken to elute analyte from the column is retention clip. It is a consider as a feature of a molecule.

The clip between sample injection and an analyte extremum making a sensor at the terminal of the column is termed theretention clip ( tR ) . It is a feature of the solute or molecule.

Rearward stage HPLC:

Rearward stage HPLC is used in pharmaceutical industries to measure up the drugs before their release.

Rearward stage HPLC consists of a polar nomadic stage and non-polar stationary stage. The silicon oxide which is present in the stationary stage modify it to non-polar by attaching with the long hydro­­­carbon ironss contain either 8 or 18 C atoms.

Rearward stage chromatography works on the rule of hydrophobic forces between the solute molecules of nomadic stage and immobilised hydrophobic ligand..in that non-polar molecules tend to organize attractive forces with hydrocarbon groups because of vander wall forces.

Non-polar compounds are less soluble in dissolvers because they need to interrupt the H bonds ­­­­because they squeeze­­­ in the H2O and methyl alcohol. So, the non-polar compounds have longer keeping clip and polar compounds have smaller keeping clip.

Applications:

1. Quantification of drugs and their metabolites in biological fluids.

2. Determination of divider coefficients and pKa of drugs and of drug protein binding.

3. The combination of hard-hitting liquid chromatography ( HPLC ) with UV/visible sensing provide a accurate, precise and robust method for quantitative analysis of pharmaceutical merchandises in the industry.

4. Monitoring of the stableness of pure drug substances and of drugs in preparations, with quantification of any debasement merchandises.

Mass spectrometry:

Mass spectrometry is an analytical technique used for dividing and placing the molecular mass of the compound by their mass to bear down ( m/z ) ratio.

This modern analytical chemical science technique is the combination of physical separation capableness of high public presentation liquid chromatography ( HPLC ) and the mass spectroscopy capableness of analyzing the mass of the compound of involvement. This technique is immensely used in drug development surveies like quantitative bioanalysis, quality control, dross designation, glycoprotein and peptide function, metabolite designation, In vivo drug testing etc. , because of its really high sensitiveness and selectivity.

The chief advantage of mass spectrometry is to place the unknown compounds by utilizing picogram ( 10-12 ) sums.

Mass spectrometer contains three cardinal constituents viz. ion souce, mass analyzer and sensor.

Applications:

* Mass spectrometry provides a extremely specific method for finding or corroborating the individuality or construction of drugs and natural stuffs used in their industry.

* Mass spectrometry has become a of import tool in proteomics, which is presently a major tool in drug find.

* Mass spectrometry in concurrence with gas chromatography ( GC-MS ) or liquid chromatography ( LC-MS ) provides a method for characterizing the drosss in drugs and preparation excipients.

* GC-MS and LC-MS provide a extremely specific and sensitive method for finding the drugs and their metabolites in biological fluids and tissues.

SOLID PHASE EXCTRACTION:

Solid stage extraction is an extraction method that uses a solid stage and liquid stage to insulate a analyte from the solution. SPE is normally used to clean up the sample before utilizing a chromatographic method to quantify the sum analyte in the sample.

Applications:

Solid stage extraction is utile for selective separation of interferants from analytes, which is non accomplishable by liquid/liquid extraction.

Solid stage extraction is widely used in bio analytical measurings and environmental monitoring for concentrating hint sums of analytes.

In clinical applications, SPE is used to supervise the drug residues in piss, blood and other organic structure fluids.

SPE is used to supervise hints of accelerates in fire dust in forensic section.

Advantages:

Small volumes of dissolvers required for both rinsing and elution.

Emulsions will non organize between two stages like liquid/liquid extraction.

Extractions can be carried out in batches instead than serially.

Rearward stage SPE:

Rearward stage SPE separate the analytes based on their mutual opposition. Reverse stage SPE involves reasonably polar or polar nomadic stage and non-polar stationary phase.the catrigade contains Due to the hydrophobic consequence the low to mild polar compounds retain on the SPE cartiage. ­­­

VALIDATION METHODS:

The term proof is defined as the procedure of set uping

“The procedure of set uping documented grounds which provides a high grade of confidence that a specific procedure will systematically bring forth a merchandise meeting pre-determined specifications and quality characteristics” .

Alternatively, proof can be considered as the documented activity performed to show that a given public-service corporation, system, procedure or piece of equipment does what it purports to make.

Therefore proof can be applied to many different phases of industry, e.g. procedure proof, cleaning proof, analytical proof.

Generally proof is applied to different types of analytical trial methods

I. Quality control trials ( for finished merchandise )

two. In procedure control methods ( for intermediate, bulk merchandise )

three. Acceptance of trial methods ( for natural stuff )

four. Methods used for mensurating the pharmaceutical substances in biological samples

v. Methods used in research and development for new medical specialty.

To guarantee that all pharmaceutical makers validate their analytical methods to the highest criterions, a set of criterions has been developed and agreed internationally for usage in the pharmaceutical industry. These have been produced by the International Conference on Harmonisation ( ICH ) which has produced guidelines in a figure of facets of pharmaceutical quality.

ICH Q2 ( R1 ) is the revised criterion for “Validation of Analytic Procedures: Text and Methodology” . Harmonizing to ICH Q2 ( R1 ) , analytical proof should be carried out by measuring the undermentioned standards ( standard singular ) of the method

* Accuracy

* Preciseness

o Repeatability

o Reproducibility

o Intermediate preciseness

* Linearity

* Specificity/selectivity

* Scope

* Sensitivity

o Limit of sensing ( LOD )

o Limit of quantification ( LOQ )

* Robustness

* Stability of analytical solution

The clip to transport out the full proof is about 4-6 hebdomads for the analyst.

Accuracy:

The truth of analytical process expresses the intimacy understanding between the value that is accepted either as a conventional true value or accepted mention value. [ 3 ]

The ICH recommends that a lower limit of nine findings over a lower limit of three concentrations are used to show the truth.

Accuracy can be expressed in footings of per centum recovery, where
% Accuracy = Mean observed concentration ten 100

Expected concentration

Preciseness:

The preciseness of an analytical process expresses the intimacy of understanding ( grade of spread ) between a series of measurings obtained from multiple sampling of the same homogenous sample under the prescribed conditions

It is by and large considered at three degrees. They are duplicability, intrinsic preciseness and repeatability.

The preciseness of an analytical method is normally expressed as the Standard Deviation, Relative Standard Deviation ( RSD ) or Coefficient of Variation ( CV ) of the method,

RSD ( CV ) = Standard Deviation x 100 %

Mean Value

Recall from statistics that 68 % of consequences in a series of measurings lie within the scope
Mean ± 1 criterion divergence

And that approx 95 % of consequences prevarications within
Mean ± 2 criterion divergences

Repeatability:

It expresses the preciseness under same operating conditions in a short interval clip to acquire the same consequences for the same batch. it besides known as intra-assay preciseness. The repeatability of analytical methods used in the QC of drugs and medical specialties should be high.

At least 5 or 6 findings of three matrices at two or three concentrations should be done.

Intermediate preciseness:

It expresses the preciseness of the analytical method obtained within a individual research lab by utilizing different operators and proving the same batch of stuffs at different times utilizing different equipments and different reagents. It is calculated as RSD.

Reproducibility:

It expresses the preciseness between the research labs. There are fluctuations like difference in humidness and temperature, fluctuation in stuff and conditions

One-dimensionality:

“The Linearity of an analytical process is its ability to obtain trial consequences that are straight relative to the concentration of analyte in the sample within a given range” .

The one-dimensionality should be measured over 0-150 % of the expected concentration of the drug. The one-dimensionality is determined by a series of 3 to 6 injections of 5 or more criterions.

The advantage of holding a relative relationship is that merely a individual criterion solution is needed to build the standardization graph — the origin sets the 2nd informations point. If a additive but non-proportional graph is obtained at least two and sooner five standard solutions are needed for the standardization graph.

For those standardization curves that are non consecutive lines ( rectilinear ) , another mathematical relationship may be used but the relationship must be demonstrated ( e.g. a quadratic or curvilineal relationship ) .

The ICH recommends for truth describing the standardization curve must incorporate correlativity co-efficient, y-intercept, incline of the arrested development line and residuary amount of squares.

Scope:

“The scope of an analytical process is the interval between the upper and lower concentrations ( or sums ) of analyte in the sample for which it has been demonstrated that the method has an acceptable grade of truth, preciseness and one-dimensionality

For assay trials ICH requires the minimal specified scope of to be 80 to 120 per centum of the trial concentration.

Robustness:

“Robustness of an analytical method is a step of its capacity to stay unaffected by little but calculated fluctuations in method parametric quantities and provides an indicant of its dependability during normal usage.”

Ideally, a method should non be sensitive to little fluctuations in temperature, pH, the concentrations or purenesss of the reagents etc. or the clip of reaction ( if a reaction is necessary )

Robustness can be measured by doing little alterations in the parametric quantities of the analytical method, like pH, flow rate, nomadic stage composing, temperature, injection volume, wavelength.

Sensitivity:

Sensitivity is the name given to that property of an analytical method or process that indicates how small of the analyte can be measured with the desired truth and preciseness.

It is of import that the everyday analytical method should be sufficiently sensitive for mensurating the analyte in the sample, e.g. intermediate preparations, and majority and finished merchandises, so that the method will be able to observe, quantitatively, any concentrations that well deviate from the specification.

ICH Q2 ( R1 ) requires sensitiveness to be evaluated in two ways
4.1 Limit of Detection

“The sensing bound ( bound of sensing, LoD ) is the lowest sum of the analyte in a sample that can be detected, but non needfully quantified as an exact value” ( ICHQ2 ( R1 )

Limit of Quantitation ( LoQ )

“The quantitation bound of an single analytical process is the lowest sum of the analyte in a sample that can be quantitatively determined with suited preciseness and accuracy” . The LoQ is higher than LoD.

Specificity:

Specificity is the ability to mensurate the analyte qualitatively and/or quantitatively without intervention from any other substance. A method that lacks specificity will endure intervention from other constituents in the sample and give consequences that inaccurate.

System suitableness trials:

System suitableness trials is used to verify the declaration, column effiency and repeatability of a chromatographic system to guarantee its adequateness for peculiar method [ .6 ]

System suitableness tests involves the comparsion of chromatographic hint with a standard hint under ideal conditions. These allows the comparing of peak form, top out breadth and baseline declaration.

The primary SST parametric quantities are declaration, repeatability, column efficiency and chasing factor. These are most of import to bespeak as they indicate system stableness, preciseness and system specificity.

System suitability trial contains the trials like

O Number of theoretical home bases ( efficiency )

O Relative criterion divergence ( preciseness )

O Capacity factor

O Separation ( comparative keeping )

O Resolution

O Tailing factor

Image [ 7 ]

Plate figure or figure of theoretical home bases:

Plate figure is the step of the acuteness of the extremums and efficiency of the column. Number theoretical home bases measured in assorted ways by utilizing peak breadth at the base and peak breadth at half of the peak tallness.

N=16 ( RT/W ) 2

Where,

N = no. of theoretical home base

RT = keeping clip

W = breadth of extremum

And

w1/2is the peak breadth at half height [ 8 ] .

If the length of the column is L, so HETP is

HETP=L/N

Where N=number of theoretical home bases and

L=length of the column

The higher the N and smaller the HETP gives better efficiency.

CAPACITY FACTOR:

Capacity facror used to depict the migration rate of an analyte on column.it is besides called as keeping factor.

Capacity factor value gives the indicant of how long the each constituent wae retained on column

Capacity factor can be calculatd by utilizing the expression

K= ( Tr–To ) /To

Where Tr=retention clip of the extremum of intrest

T0=retention clip of the unretained extremum

When the analyte capacity factor is less than one, elution is so fast that accurate finding of keeping clip is hard and retention factor more than 20 menas elution takes long clip.

Ideally capacity factor is in between 1 to 5.

SEPARATION FACTOR:

Separation factor describes the separation of two extremums on column. It is calculated by utilizing capacity factor because peak separation is depends upon the constituents interaction with stationary stage.

If we consider the A and B as two extremums, so

Separation factor ( ? ) =KB/KA

Where, KA =capacity factor for Angstrom

KB=capacity factor for Angstrom

When A elutes faster than B. the separation factor is ever greater than one.

PEAK RESOLUTION:

Peak declaration is used in separation between extremum and efficiency of column. Peak declaration is express ratio of distance between the two extremums to the average value of breadth of extremum.

And peak declaration is calculated by a undermentioned equation.

RS = 2 ( TR2 – TR1/ W2 + W1 )

Where, TR2 = keeping clip of your late eluting extremum

TR1 = keeping clip of your early eluting extremum

W2 = breadth of your late eluting extremum

W1 = breadth of your early eluting extremum

Base line declaration is achieved when R=1.5

Shadowing Factor:

This is a step for the dissymmetry of the extremum.

It is defined as the distance from the front incline of the extremum to the back incline divided by twice the distance from the halfway line of the extremum to the forepart incline, with all measurings made at 5 % of the maximal extremum height..

Tf=

RELATIVE STANDARD DEVIATION:

Preparation of sample:

Preparation of nomadic stage:

To a conelike flask add 820 milliliter of H2O and 170 milliliter of methyl alcohol,

RESULTS AND DISCUSSION:

One-dimensionality:

Num.

Con

( µg/ml )

Peak Area

Mean

South dakota

% RSD

1

10

1st tally – 298.133

2nd tally – 237.803

3rd tally – 297.335

4th run-237.837

5th run-237.417

2

20

1st tally – 497.8

2nd tally – 497.472

3rd run – 497.871

4th run-497.381

5th run-497.99

3

30

1st tally – 715.5

2nd tally – 714.536

3rd tally – 785.163

4th run-716.08

5th run-716.679

4

40

1st tally – 969.99

2nd tally – 969.293

3rd tally – 969.385

4th run-968.694th

5th run-969.473

5

50

1st tally – 1223.124

2nd tally – 1223.599

3rd tally – 1222.733

4th run-1224.325

5th run-1226.906

6

70

1st tally – 1473.126

1472.895nn

2nd tally – 1472.75

3rd tally – 1475.189

4th run-1552.561

5th run-1474.450tht

7

?80

1strun – 1800.511­­

2ndrun– 1729.729

3rdrun – 1802.75

4th run-1802.158

5th run-1801.612

6th run-1800.917

7th run-1801.515

8th run-1801.636

9th run-

10th run-1802.717

10th run-

Entire

2.6896

Residual analysis statical out put of standard Theobromine from HPLC-UV:

Standard theobromine arrested development analysis from HPLC-UV is given below.

SUMMARY OUTPUT

Arrested development Statisticss

Multiple R

0.996853

R Square

0.993716

Adjusted R Square

0.993541

Standard Error

43.07845

Observations

38

Analysis of variance

Df

United states secret service

Multiple sclerosis

F

Significance F

Arrested development

1

10563775

10563775

5692.448

3.1E-41

Residual

36

66807.1

1855.753

Entire

37

10630582

Coefficients

Standard Error

T Stat

P-value

Intercept

68.57384

15.30126

4.481582

7.24E-05

10

22.1743

0.293901

75.44831

3.1E-41

RESIDUAL OUTPUT

Observation

Predicted Y

Remainders

Standard Remainders

1

290.3168

-52.5138

-1.23584

2

290.3168

7.018187

0.165164

3

290.3168

-52.4798

-1.23504

4

290.3168

-52.8998

-1.24493

5

512.0598

-14.2598

-0.33558

6

512.0598

-14.5878

-0.3433

7

512.0598

-14.1888

-0.33391

8

512.0598

-14.6788

-0.34545

9

512.0598

-14.0698

-0.33111

10

733.8028

-18.3028

-0.43073

11

733.8028

-19.2668

-0.45342

12

733.8028

51.36024

1.208695

13

733.8028

-17.7228

-0.41708

14

733.8028

-17.1238

-0.40298

15

955.5457

13.14827

0.309427

16

955.5457

11.44427

0.269326

17

955.5457

13.74727

0.323524

18

955.5457

13.83927

0.325689

19

955.5457

13.92727

0.32776

20

1177.289

45.8353

1.078673

21

1177.289

46.3103

1.089851

22

1177.289

45.4443

1.069471

23

1177.289

47.0363

1.106937

24

1177.289

49.6173

1.167677

25

1399.032

106.5703

2.50799

26

1399.032

29.48633

0.693921

27

1399.032

32.83933

0.77283

28

1399.032

109.4913

2.576732

29

1399.032

34.09233

0.802317

30

1842.518

-42.0066

-0.98857

31

1842.518

-42.7886

-1.00697

32

1842.518

-39.7676

-0.93588

33

1842.518

-40.3596

-0.94981

34

1842.518

-40.9056

-0.96266

35

1842.518

-41.6006

-0.97901

36

1842.518

-41.0026

-0.96494

37

1842.518

-40.8816

-0.96209

38

1842.518

-39.8006

-0.93665

Residual analysis of standard Theobromine:

Calibration curve:

The standardization curve calculated by arrested development analysis utilizing the equation

Y=mx+c,

Where Y= response of center selected on the standardization curve,

m = average incline coefficient,

ten = the concentration for the standardization line equation,

hundred = y- intercept for average swill coefficient

by puting the values in above equation,

hypertext transfer protocol: //web1.caryacademy.org/chemistry/rushin/studentprojects/compoundwebsites/2003/theobromine/properties.htm

hypertext transfer protocol: //maxshouse.com/Drugs_To_Avoid_in_Cats.html

hypertext transfer protocol: //www.labcompliance.com/tutorial/methods/default.aspx # 03_standard

. Gurudeep Chatwal, Sham K.Anand ( 2007 ) . Instrumental Methods of Chemical Analysis. 5th erectile dysfunction. India: Himalaya Publishing House. p 2.270-2.302

Solid stage extraction: hypertext transfer protocol: //www.springerlink.com/content/h72xx3624q122085/fulltext.pdf hypertext transfer protocol: //www.chemistry.adelaide.edu.au/external/soc-rel/content/spe.htm

Rps-http: //www.chemguide.co.uk/analysis/chromatography/hplc.html

Validation: 6 ) Michael Dong, Roy Paul, and Lea Gershanov. ( sepetember 2001 ) . Geting the extremums perfect: System suitableness for HPLC On-line testing can guarantee informations quality in pharmaceutical assays..todays ‘s chemical science at work. 10 ( 01 ) , 38-40,42.

7. hypertext transfer protocol: //www.sciencedirect.com/science? _ob=ArticleURL & A ; _udi=B6TG8-477FPW5-2 & A ; _user=7833342 & A ; _rdoc=1 & A ; _fmt= & A ; _orig=search & A ; _sort=d & A ; _docanchor= & A ; view=c & A ; _searchStrId=1111432776 & A ; _rerunOrigin=google & A ; _acct=C000027518 & A ; _version=1 & A ; _urlVersion=0 & A ; _userid=7833342 & A ; md5=167d3d4e5da6a8170e621eb63a212246

8. hypertext transfer protocol: //teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm hypertext transfer protocol: //www.microsolvtech.com/calculators_chrom.asp #

R.A. new wave 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 debut to analytical method proof

By – 05/20/2007

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