HPTLC - instrumentation, principle, methods of development, validation methods, applications.... useful for the analysis of drugs

1. HPTLC
METHOD DEVELOPMENT, VALIDATION AND
ANALYSIS OF BOTANICALS
PREPARED BY
GOWTHAMRAJ.S
M.Pharm – I – Year
Department of Pharmaconosy

2. CONTENTS
 Introduction
 Principle of HPTLC
 General methodology for HPTLC
 Validation of developed method
 Fingerprint analysis of botanicals
 Applications

3. INTRODUCTION
 It is also known as planer or flat bed chromatography.
 It is the automated, advanced, sophisticated form and improved method of TLC.
 HPTLC is conducted on TLC plates which are coated with purified silica gel with a
particle range of 2-10um as opposed to 2-25um for standard commercial TLC
plates.
 The narrow particle size range means that a greater number of theoretical plates
are available for separation and thus the spots on the TLC plate remain tighter.
 These type of plates may be run in a standard type of TLC tank but optimal
performance is obtained from horizontal development of the plates using HPTLC.
 High Performance Thin Layer Chromatography (HPTLC) is the most powerful
advanced form of Thin Layer Chromatography (TLC) and consists of
chromatographic layers of utmost separation efficiency and the application of
sophisticated instrumentation for all steps in the procedure include accurate
sample application, standardized reproducible chromatogram development and
software controlled evaluation

4. PARAMETER TLC HPTLC
Technique Manual Instrumental
Efficiency Less High
Layer Lab made/ pre- coated Pre coated
Mean particle size 10-12 um 5-6 um
Layer thickness 250 um 100 um
Plate height 30 um 12 um
Solid support Silica gel, alumina, kiesulguhr Silica gel – normal phase
c8 and c18 – reverse phase
Sample spotting Manual spotting Auto sampler
DIFFERENCE B/W TLC AND HPTLC TECHNIQUES:

5. PRINCIPLE
 HPTLC involves the similar theoretical principle of TLC i.e. the principle of
separation is adsorption
 An analyte migrates up or across a layer of stationary phase ( most commonly
silica gel), under the influence of a mobile phase , which moves through the
stationary phase by capillary action.
 The distance moved by the analyte is determined by its relative affinity for the
stationary vs the mobile phase.
 Mobile phase flow by capillary effect and component move according to their
affinities towards the adosrbant.
 The component with higher affinity towards the adsorbant travels slowly.
 The component with lower affinity towards the stationary phase travels faster.
 Finally the components are separated on a chromatographic plate according to
their affinity and separation also based on their solubility in mobile phase.

6. INSTRUMENTATION

7. GENERAL METHEDOLOGY INVOLVED IN HPTLC
Selection of chromatographic plates
Layer washing
Activation of pre-coated plates
Sample preparation and applications
Selection of mobile phase
Pre- conditioning
Chromatographic development and drying
Detection and visualization
documentation

9. 1. SELECTION OF CHROMATOGRAPHIC PLATES:
Pre-coated plates : The plates with different support materials and sorbent layers
with different format and thickness are used for qualitative and quantitative
analysis.
Supported materials used in plates
 Glass
 Polyester/polyethylene
 Aluminium
Sorbents used in plates
 Silica gel 60f
 Aluminium oxide
 Cellulose
Silica gel chemically modified
a) amino group (NH2)
b) b) CN group.
Smaller particle size of silica helps in greater resolution and sensitivity.

10. 2. LAYER PRE – WASHING
 The main purpose of the pre-washing is to remove impurities which include
water vapours and other volatile substances from the atmosphere when they get
exposed in the lab environment.
Methods
 Ascending
 Dipping
 Continuous
Solvents
 Methanol
 chloroform : methanol (1:1)
 Chloroform : methanol: ammonia (90:10:1)
3. SAMPLE PREPARATION
 Sample and reference substances should be dissolved in the same solvent to
ensure comparable distribution at starting zones.
 It needs a high concentrated solution, as very less amount of sample need to be
applied and dry the plates and store in dust free atmosphere.

11. Solvents
 Methanol,
 chloroform : methanol (1:1)
 Chloroform : methanol: ammonia (90:10:1)
 Usual concentration range is 0.1-1 ug / ul , above this causes poor separation
and volume recommended for HPTLC – 0.5-5ul.
 The size of sample spot applied must not exceed 1mm in diameter.
 Capillary tubes, micro bulb pipettes, micro syringes, automatic sample
applicator are used.

12. 4.SELECTION OF MOBILE PHASE
 Chemical properties of analytes and sorbent layer factors should be considered
while selection of mobile phase.
 Various components of mobile phase should be measured separately and then
placed in mixing vessel.
 The less amount of mobile phase is required then TLC
 This prevents contamination of solvents and also error arising from volumes
expansion or contraction on mixing
 Multi component mobile phase once used not recommended for further use due
to different evaporation and adsorption by layer.
5.PRE- CONDITIONING (CHAMBER SATURATION)
 Un saturated chamber causes high Rf values.
 Saturated chamber by lining with filter paper for 30min prior to development
uniform distribution of solvent vapour less solvent for the sample to travel
lower Rf values.
 Chamber saturation influence separation profile.

13. 6.CHROMATOGRAPHIC DEVELOPMENT AND DRYING
Various forms of chromatographic development like
 Ascending
 Descending
 Horizontal
 Continuous
 Gradient
 Multidimensional
 For HPTLC plates, migration distance of 5-6 mm is sufficient.
 After development, plates are removed from the chamber and dried to remove
traces of mobile phase.

14. PROBLEMS ENCOUNTERED DURING CHROMATOGRAPHIC
DEVELOPMENTS
Tailing :
This may occur due to the presence of traces of impurities or more than one ionic
species of substance under chromatography.
This can be reduced by buffering the mobile phase system with acidic (1-2 % acetic
acid) or basic ( ammonia ) solution. It keeps the materials to be separated in non-
ionic forms.
Diffusion:
This is seen as zones on chromatographic plates. This may arise due to non-
uniformity of mobile phase, longitudinal diffusion between mobile phase and
stationary phase or due to non-equilibrium of stationary phase.

16. 7.DETECTION AND VISUALIZATION
 Detection under UV light is first choice
 Non destructive and spots of fluorescent compounds can be seen at 254nm short
wave length or at 366nm long wave length.
 Spots of non fluorescent compounds can be seen fluorescent stationary phase is
used – silica gel GF
 Non UV absorbing compounds like ethambutol, dicycloamine dipping the plates in
0.1% iodine solution.
8.SCANNING AND DOCUMENTATION
 The development of HPTLC plates are scanned at selected UV regions wavelength
by the instrument and the detected spots are seen on computer in the form of
peaks.
 The scanner converts band into peaks and peak height or area is related to the
concentration of the substance on the spot.
 The peak height and area under the spot (curves) are measured by the
instruments and are recorded as per cent on the printer.

17. ADVANTAGES
 Analysis of substances in complex matrices like plant materials, lipid samples,
sample with high sugar content.
 HPTLC fingerprint of herbal drug samples visually either confirms or rejects
the plant identity.
 HPTLC is used for multiple detection methods on the same sample and plate,
UV/VIS/fluorescence/hyperspectral/derivatization or effected directed. In
contrast to other chromatographic techniques, the separated analytes of the
sample remain on the plate.
 Analysis of multiple samples in parallel without cross contamination.
 HPTLC is used for purity control of chemicals, pesticides, steroids, and water
analysis.
 Low running and maintenance costs and disposable layer
 Sample and standard both can be used at a time.
 Efficient data acquisition and processing

18. HPTLC DEVELOPMENT METHOD

19. VALIDATION OF DEVELOPED METHOD
1. Simple and precise HPTLC methods were developed for the simultaneous
estimation of two anti-inflammatory drugs (curcumin and galangin). The method
was tailored to analyze both drugs in their commercial dosage form (capsules) with
no interference from ingredients. Chromatographic separation was performed over
pre-coated TLC plates (60 F254, 20 cm × 10 cm, 250µm thickness, Merck,
Darmstadt, Germany) via a linear ascending technique using n-hexane, ethyl
acetate, acetic acid, and methanol as the mobile phase. Detection and
quantification was achieved at 404 nm through spectro densitometric analysis
2. The report of TLC densitometric method, which has been developed and validated
for quantification of stigmasterol from petroleum ether extract of leaves and stems
of Bryophyllum pinnatum. The separation was performed on TLC aluminum plates
precoated with silica gel 60 F254. Good separation was achieved in mobile phase
using Chloroform : Ethanol (9.8:0.2 v/v). Determination and quantitation were
performed by densitometric scanning at 490 nm in reflection/absorbance mode

20. precise and accurate HPTLC method for its estimation as bulk and in tablet dosage
form. The chromatographic separation was carried out on pre-coated silica gel 60
F254 aluminium plates using mixture of methanol and toluene (4:3%v/v) as mobile
phase and densitometric evaluation of spots were carried out at 235nm
Lipophilic C-18, C-8, C-2; phenyl chemically-modified silica gel phases; and
hydrocarbon- impregnated silica gel plates developed with a more polar aqueous
mobile phase, such as methanol–water or dioxane–water, are used for reversed-
phase TLC.
1. A simple, precise, accurate and high performance thin layer chromatographic
method has developed and validated for the estimation of Olmesartan medoxomil
and hydro-chlorthiazide simultaneously combined dosage forms. The stationary
phase used is precoated silica gel 60F254. the mobile phase was a mixture of
acetonitrile: chloroform: glacial acetic acid (7:2:0.5, v/v/v). The detection of spots
was carried out at 254nm

21. 2. A simple, precise, accurate and rapid high performance thin layer
chromatographic method has been developed and validated for the estimation of
tenoxicam in the micro emulsion gels. Tenoxicam was chromatographed on silica gel
60 F254 TLC plate, as a stationary phase. The mobile phase was toluene: ethyl
acetate: formic acid (6:4:0.3 v/v/v) used
3. A simple, precise, specific and accurate high performance thin layer
chromatographic method has been developed for the simultaneous determination of
Cinitapride and Omeprazole in pharmaceutical dosage form. The separation was
carried out on Merck HPTLC aluminum plates of silica gel G60 F254, (20 × 10 cm)
with 250μm thickness using chloroform: ethyl acetate: methanol (7.3: 2: 0.7, v/v/v)
as mobile phase. HPTLC separation of the two drugs followed by densitometric
measurement were carried out in the absorbance mode at 277 nm
It describes developed and validated thin layer liquid chromatography (TLC) method
for the simultaneous estimation of telmisartan and ramipril in a combined dosage
form.

22. 4. An accurate, sensitive, precise, reliable, and quick method for the determination of
cholesterol content by high-performance thin layer chromatography is developed. In this
method, aluminum-backed pre-coated silica gel 60 F254 plates were used as the
stationary phase and the samples were sprayed with the help of CAMAG sample
applicator Linomat. The chromatogram was developed with the mobile phase consisting
of chloroform: methanol (9.5:0.5, v/v)
5. A simple, precise and accurate HPTLC method Duloxetine Hydrochloride for its
estimation as bulk and in tablet dosage form. The chromatographic separation was
carried out on pre-coated silica gel 60F254 aluminium plates using mixture of
Chloroform: methanol (8:1 v/v) as mobile phase and densitometric evaluation of spots
was carried out at 235nm

23. VALIDATION OF DEVELOPED METHOD OF TRANDOLAPRIL
• A simple, precise, accurate and rapid high performance thin layer
chromatographic method has been developed and completely validated for the
estimation of trandolapril in bulk and pharmaceutical dosage forms.
• Quantification of trandolapril was carried out with percolated silica gel 60F 254 as
stationary phase using mobile phase consisting of Chloroform: Methanol: Acetic
acid (8:1.5:0.5 v/v/v) and scanned in Absorbance/Reflectance mode at 212 nm
using Camag TLC scanner 3 with WinCATS software.
• The Rf value of trandolapril was found to be 0.54 (±0.03). The proposed method
has permitted the quantification of trandolapril over the linearity range of 25- 150
ng/spot and its percentage recovery was found to 99.7%.
• The intra day and inter day precision were found to be 1.26% and 1.4%,
respectively.
• The limit of detection and the limit of quantification were found to be 18 ng/spot
and 54 ng/spot, respectively.

24. • The proposed method can be successfully applied for the estimation of drug
content of different marketed formulations simultaneously on a single plate and
provides a faster and cost effective quality control tool for routine analysis of
trandolapril as bulk drug and in tablet dosage forms.
TRANDOLAPRIL
• Trandolapril, chemically, it is (2S, 3aR, 7aS)-1-[(S)-N-[(S)-1-carboxy-3-
phenylpropyl] alanyl] hexahydro-2- indolinecarboxylic acid, 1-ethyl ester [1] and is
not official in any pharmacopoeia.
• Trandolapril is an orally administered angiotensin converting enzyme inhibitor
that has been used in the treatment of patients with hypertension and congestive
heart failure, and myocardial infarction. Literature survey revealed that few HPLC
methods were reported for the estimation of trandolapril in the biological fluids.
The present study illustrates development and validation of a simple, accurate,
precise and specific HPTLC method for the estimation of trandolapril tablet dosage
forms.

25. EXPERIMENTAL
REAGENTS
Pure working standard of trandolapril was procured as a gift sample from
Ranbaxy Ltd., Himachal Pradesh. All chemicals and reagents used were of analytical
grade. A Silica gel 60F 254 TLC pre coated aluminum plates (10×10 cm, layer
thickness 0.2 mm, E. Merck, Mumbai) were used as a stationary phase. Chloroform:
Methanol: Acetic acid (8:1.5:0.5 v/v/v) was used as mobile phase and methanol was
used as solvent.
APPARATUS
A CAMAG HPTLC system (Switzerland) comprising a CAMAG Linomat IV
semiautomatic sample applicator, a CAMAG TLC Scanner 3, A CAMAG twin-trough
chamber (10 × 10 cm), CAMAG CATS 4 software, A Hamilton syringe (100 µl), A
Shimadzu libror AEG-220 weighing balance and A ultra sonicator (Frontline FS-4,
Mumbai) was used during the study

26. CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions were optimized and estimations were
performed on a stationary phase, pre coated silica gel 60 F254 aluminum sheets
(10×10 cm) which were pre-washed with methanol and dried in air, with mobile
phase of Chloroform: Methanol: Acetic acid (8:1.5:0.5 v/v/v) . The chromatographic
chamber and plate was allowed to saturate for about 30 min and the migration
distance allowed was 72 mm. The wavelength scanning was performed at 212 nm
keeping the slit dimension 5×0.45 mm. The source of radiation was deuterium lamp
emitting a continuous UV spectrum between 190-400nm. The standard solutions of
trandolapril was spotted and developed at constant temperature of 25 ± 2ºC.
PREPARATION OF MOBILE PHASE
Chloroform: Methanol: Acetic acid (8:1.5:0.5 v/v/v) was employed as
mobile phase.
PREPARATION OF STANDARD SOLUTION OF TRANDOLAPRIL
A working standard of trandolapril about 2.5 mg was accurately weighed
and transferred in to 100 ml volumetric flask.

27. A volume of methanol about 25 ml was added and sonicated for about 20 min; finally
the volume was made up to 100ml with methanol to obtain the concentration about 25
µg/ml. From this stock solution 0.1 ml was taken and the volume made up to 100ml to
get concentration about 25ng/ml.
PREPARATION OF CALIBRATION CURVE
Aliquots (1, 2, 3, 4, 5 and 6 µl) of standard solution of trandolapril were
spotted on pre coated TLC plates using semi automatic spotter under nitrogen stream.
The plate was dried in air and developed up to 72 mm at constant temperature with a
mixture of Chloroform: Methanol: Acetic acid (8:1.5:0.5 v/v/v) as mobile phase in a
CAMAG twin through chamber which was previously saturated with mobile phase for
about 30 min. the plate was removed from the chamber and dried in air. Photometric
measurements were performed at 212 nm in absorbance/reflectance mode with the
CAMAG TLC scanner 3 using CATS 4 software incorporating track optimizing option.
The standard plot of trandolapril was established by plotting the peak area Vs
concentration (ng/ml) corresponding to each spot.

28. METHOD DEVELOPMENT
Trandolapril was soluble in methanol, there fore methanol was selected as the
solvent. A solvent system consisting of Chloroform: Methanol: Acetic acid (8:1.5:0.5
v/v/v) was selected as mobile phase, that would give dense and compact spot with
appropriate Rf values was selected for quantification of Trandolapril in
pharmaceutical formulations. The present HPTLC method for the quantification
trandolapril in bulk and pharmaceutical dosage, revealed as simple, accurate and
precise with R f value of 0.54.

29. VALIDATION OF METHOD
• The Linearity for the detection of trandolapril was 25-150 ng/ml with R2= 0.998;
Y=21.07x + 21.71. The results were shown in the Table-1. The precision of the
method (System reproducibility) was assessed by spotting 3 µl of drug solution six
times on a TLC plate, followed by development of plate and recording the peak area
for 6 spots. The % RSD for peak area values of trandolapril was found to be 1.04%.
• The method reproducibility (The intra-day precision) was determined by analyzing
standard solutions in the concentration range of 75 ng/spot to 100 ng/spot of drug
for 3 times on the same day and inter-day precision was determined by analyzing
corresponding standards daily for 3 day over a period of one week.
• The intra-day and inter-day coefficients of variation (%RSD) are in range of 0.39 to
1.26 and 0.17 to 1.4, respectively.
• Recovery studies were carried out to assess accuracy of the method. These studies
were carried out at three levels. The percentage recovery was found to be within the
limits. The assay for the marketed formulation was established with the present
chromatographic conditions developed and it was found to be more accurate and
reliable.

30. • The average drug content was found to be 99.15% of the labelled claim.
• Limits of Detection (LOD) and Quantification (LOQ), the limits of detection and
quantitation were calculated by the method based on the standard deviation of
response (σ) and the slope of calibration plot (S), using the formulae LOD = 3.3σ/S
and LOQ = 10σ/S. The LOD and LOQ were calculated and found to be 18 ng/Spot
and 54 ng/Spot, respectively.
• Robustness was determined by altering chromatographic conditions like mobile
phase composition, Amount of mobile phase, Plate treatment, Time from spotting
to chromatography and time from chromatography. The low value of % RSD
indicates robustness of the method
• Specificity test of the proposed method demonstrated that there were no
interference form excipients. Furthermore, well shaped peaks indicate the
specificity of the method.

35. FINGERPRINT ANALYSIS OF BOTANICALS
FINGER PRINTING
 A pattern or an impression which is highly specific enough to become useful as
identifier for that particular entity.
 finger print analysis is the most potent tool for quality control of herbal
medicines because of its accuracy and reliability.
 fingerprinting is a process that determines the concentrations of a set of
characteristic chemical substance in an herb.
 it can serve as a tool for identification, authentication and quality control of
herbal drugs.
 based on the conception of phytoequivalence, the chromatography
fingerprinting and DNA fingerprinting of herbal medicines should be utilized for
addressing the problem of quality control of herbal medicines.

37. CHROMATOGRAPHIC FINGERPRINTING
• It is the most powerful approach for the quality control of herbal medicines.
• It is the chromatographic pattern produced from extract of some common
chemical components which may be pharmacologically active or have some
chemical characteristics.
• This chromatographic profile should be featured by the fundamental attributions
of integrity and fuzziness and differences so as to chemically represent the herbal
medicines investigated.
• This technique is employed for identification and authentication as well as for
determination of various adulterants and contaminants and for standardization
purpose.
 Thin layer chromatography
 High performance thin layer chromatography
 High performance liquid chromatography
 Gas chromatography
 Other hyphenated techniques

38. HPTLC FINGER PRINTING
 HPTLC is the common fingerprinting method mainly used to analyse the
compounds with low or moderate polarities.
 It is widely used for quality control of herbs and health products, identification
and detection of adulterants, substituents in the herbal products and also helps
in the identification of pesticides contents and mycotoxins.
 It is a pattern on TLC plate of separated compounds, generated according to
their highly specific Rf values, presented in sample, specific enough to become
an authentic statement or bio-chemical marker for that sample and capable
enough to provide unique, specific and characteristic identity.

39. IMPORTANCE OF HPTLC FINGERPRINTING FOR BOTANICALS
 The traditional methods are poor, time consuming and less scientific, so there is
a need to used emerging technological knowledge and sophisticated analytical
methods.
 HPTLC provide a deep inside in to the plants compound profile and their
chemistry
 There is no substitution of qualitative visual results of HPTLC for botanicals
 As a comparative with HPLC/GC and other advanced chromatographic/
molecular techniques, the HPTLC fingerprinting is cheap, fast and solvent saving
 Sensitivity and accuracy is always a matter but HPTLC is advanced enough for
providing a reliable authentic biochemical integrity statement for botanicals

40. FACTS ON MOBILE PHASE OPTIMIZATION FOR HPTC FINGERPRINTING
OF BOTANICAL
• Similiar substance = same Rf value
• Exchange the solvents o modify their rations, depends on experimental needs
• Change in solvent strength, change the Rf values
• Change in the selectivity, change in the relative positions
• Same polarity index of solvents = same Rf
• Same selectivity so similar elution order of compounds
• Achievement of optimization of mobile phase = Rf value between 0.3 to 0.5

41. PROCEDURE FOR HPTLC PLANT FINGERPRINTING
SAMPLE PREPARATIONS
• Drying – shade drying for 15 – 21 days
• Grinding – depends up on samples
• You can use soxhlet and other advanced procedure, depends up on the
experimental needs
• Selection of solvent – depends up on experimental needs ( methanol or water is
used)
EXTRACTION PREPARATIOS
• Sample : solvent = 1 : 10 respectively
• Initially 500mg powdered sample in to 10 ml solvent
• You can also make 5% HCL / solvent only / 5% NH3 modifications in the 500mg
in to 10ml solvent sample

42. MOBILE PHASE OPTIMIZATION
STEP 1
• 7 to 12 net solvents for 1-8 selectivity groups
Results
• Suitable RF value
• Too much RF value ( above 0.7 to 0.8) = then exchange solvent
• Too low RF value ( below 0.2 ) = exchange with high polarity solvents
STEP 2
• Dilution with hexane for down the high Rf
• Polar modification with acids or ammonia for increase the lower Rf
STEP 3
• Two solvents combination of different selectivity groups ratio of 1 : 1
• Short cut shunt - solvent of high Rf and solvent of low Rf can be directly
combine with each other.
• [ ratio = 1 : 1 or 10% high Rf solvent in to low Rf solvent, if results are good then
go direct combination of two solvents]

43. STEP 4
1. Improve the band shape by the use of modifiers
2. If tailings occurs then use 0.5% or 10% water
3. Miscible acid/ base can be also be exchangeable
4. Finally 2 – d chromatography for stability testing

44. STEPS INVOLVED HPTLC PLANT FINGERPRINTING
1. Plate is checked before using it for sample applicator under the 254nm, whether it
was giving fluorescence or not
2. Mark the limit of run at 80mm and direction by a HB – pencil
3. Cut all 20 x 20 cm plate, into 10x20 cm or 10x10cm plate
4. Working program is generated with the use of win CATS software in the computer
5. Specific volume of sample is taken by the use of 100 ul or 500ul Hamilton syringe
and applied on plate as the predefined 6 mm or 8mm band length by the means of
sample applicator
6. After the completion of the sample applicator program the plate is subjected for
drying with the use of a drier and then placed on to TLC plate heater for 10
minutes to remove any water or moisture content from the plate.
7. An optimized mobile phase is used for analytical demand of experiment
8. The mobile phase is subjected to the development chamber

45. 9. A filter paper rinsed with mobile phase is also subjected in the chamber for a
uniform vapo saturation of the chamber prior adding of the sample applied plate
10. Chamber saturation time is now optimized ( 20 – 25 mins)
11. The plate is placed in it, till the solvent front reached up to the distance of
80mm
12. After it, densitometry evaluation can be carried out under 254nm and 366mm in
the different – different files area calculation
13. In the last step the plate is derivatized with specific derivatizing agent for visual
identification in the derivitaizating chamber and air dried
14. Final images are quickly captured by the pate visualizer under visible white light
and fluorescence (366nm). Then densitometry scanning was performed at 540nm (
W lamp) and 366nm (Hg lamp)
15. After it , spectral analysis, Rf values calculations, peak areas calculations are
analysed for final interpretation and HPTLC finger printing pattern profile
generations

47. APPLICATIONS OF HPTLC
Pharmaceutical industries:
• Quality control
• identity purity test
• Content uniformity test
• Stability test
Food analysis:
• Quality control
• additives
• pesticides
• stability testing
Clinical applications:
• Metabolism studies
• drug screening
• stability testing

48. Cosmetics
• Identity of raw material
• Preservatives
• colouring materials
• Screening for illegal substances
Herbal medicines and botanical dietary supplements
• Identification
• Detection of adulteration
• Assay of marker compounds
Industrial applications
• Process development and optimization
• Process monitoring
• Cleaning validation
Forensics
• Detection of document forgery
• Investigation of poisoning
• Dyestuff analyses

49. QUANTITAIVE DETERMINATION
Biochemical research / biotechnology
• separation of gangliosides
Clinical
• Inorganic and organic mercury in water & human serum.
• Caffeine in urine
Cosmetics
• Hydrocortisone and cinchocaine in lanolin ointment
Environmental analysis
• Pesticides in drinking water
• Selenium in water
Natural products
• Glycosides in herbal drugs
• Glycyrrhizic acid in liquorice
Doping analysis
• Atenolol in urine

50. FINGER PRINT ANALYSIS
• HPTL finger print of valerian
• Finger print of garlic, ashwaganda
• Finger prints for identification of liquorice, ginseng.
Identification and separation of phenyl thiohydantoin – amino acid
Analysis of drugs in blood
• Seperation of phenothiazide drugs like : chlorpromazine, acetophenazine,
perphenazine, trilfuperazine and thoridazine
Identification of mycotoxins in admixture
• Detection of sterigmatocystion, zearalenone, citrinin, ochrotoxin A, patulin,
penicillic acid.
Determination of polycyclic aromatic hydrocarbons in particulate sample.
• Determination ochryesene, pyrene, fluoronthene.

51. REFERENCE
1. Ramakrishna V.S.N, Vishwottam N.K, Shrivastava W, Koteshwara M.
Quantification of trandolapril and its metabolite trandolaprilat in human plasma
by liquid chromatography/tandem mass spectrometry using solid-phase
extraction, Rap. Comm. Mas. Spec. 2006; 20:3709 – 3716.
2. Jinu John., Ankit Reghuwanshi., Usha K. Aravind., Aravindakumar,
C.T.Development and validation of a HPTLC method for thedetermination of
cholesterol concentration.Journal of food and drug analysis.2015, 2 3.219 -224.
20.
3. Kathirvel,S., Rajendra Prasad,K., and MadhuBabu,K.Development and validation
of HPTLC method for the determination of mycophenolate mofetil in bulk and
pharmaceutical formulation. Pharm Methods. 2012. 3(2): 90–93. 21.
4. Suneela S. Dhaneshwar., Deshpande, p., Patil,M., Vadnerkar,G., Dhaneshwar,
S.R., Development and validation of HPTLC method for the estimation of
Duloxetine Hydrochloride in bulk drug and in tablet dosage form.Indian Journal
of Pharmaceutical Sciences. 2008, 233-238
5. Chandel, S., Barhate, C. R., Srivastava, A. R., Kulkarni, S. R. and Kapadia, C. J.,
Development and validation of HPTLC method for estimation of Tenoxicam and its
formulations.Indian Journal of Pharmaceutical Sciences. 2012, 36-40
6. Budavari S. The Merck Index , an encyclopedia of chemicals, drugs, and
biologicals. Merck Research laboratories, New York, NY. 2001; 13 th ed: pp 9644.

52. 7. Chatwal GR, Aanad SK. Instrumental methods of chemical analysis. 5th ed.
Mumbai: Himalaya Publishing House; 2002
8. Beckett AH, Stenlake JB. Practical pharmaceutical chemistry. 4th ed. Part 2,
New Delhi: CBS Publishers and Distributers; 2001. p. 116-67..
9. Udhan RR, Jaybhaye S, Pathan IB. Development and validation of a simple UV
spectrophotometric method for the determination of desloratadine both in bulk
and marketed dosage formulation. Int J Pharm Res Sci 2013;1(1):33-6
10.International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceutical for Human Use. Validation of Analytical
Procedures. Text and Methodology. Geneva, Switzerland.2001