Instrumental Thin-Layer Chromatography || Pharmaceutical Applications of High Performance Thin Layer Chromatography

C H A P T E R

17

Pharmaceutical Applicationsof High Performance Thin Layer

Chromatography

Sunil R. Dhaneshwar 1, 21Department of Pharmaceutical Chemistry, RAK College of Pharmaceutical

Sciences, Ras Al Khaimah Medical and Health Sciences University,Ras Al Khaimah, UAE; 2 Poona College of Pharmacy, Bharati Vidyapeeth

University, Erandwane, Pune, India

17.1 INTRODUCTION

The introduction of high performance thin layer chromatography(HPTLC) in pharmaceutical analysis represents an important milestonehaving superseded conventional TLC where it has several distinctadvantages. Before we discuss the pharmaceutical applications of HPTLCit would be appropriate to highlight these advantages, as it will help usunderstand why HPTLC is becoming more popular amongst pharma-ceutical analysts.

1. Being an open method, sitting substances (spots which do notmove from the point of application) can be identified easily andcorrective action taken (e.g., indicates the need for a differentmobile phase or stationary phase).

2. Since development and detection are separate steps,ultraviolet (UV) absorbing mobile phases do not cause anyproblem.

3. Samples can be detected by either universally applied stainreagents, such as iodine vapors, or specifically applied reagent for aparticular sample, such as ninhydrin for amino acids or2,4,6-trinitrophenylhydrazine for ketones.

Instrumental Thin-Layer Chromatography

http://dx.doi.org/10.1016/B978-0-12-417223-4.00017-0 451 Copyright � 2015 Elsevier Inc. All rights reserved.

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17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE452

4. A large number of samples (e.g., 30e40) can be applied to a singleTLC plate and separated simultaneously.

5. Since HPTLC uses disposable stationary phases, time and solventconsuming column cleanup procedures are avoided.

6. Simultaneous analysis of samples and standards under identicalexperimental conditions is possible. This is a unique feature ofHPTLC which is not possible with any other form ofchromatography.

7. Choice of solvent for sample preparation is not very critical as thesample solvent and mobile phase do not come in contact with eachother.

8. Mobile phase can be prepared with analytical grade solvents andnot chromatographic grade solvents. Moreover no prior treatmentsuch as filtration or degassing is required.

9. Different volumes of standard or sample solutions can be appliedon the same TLC plate. Thus it is possible that during a single run,construction of a calibration curve and sample quantification canbe achieved simultaneously.

10. All the commonly used stationary phases of High PerformanceLiquid Chromatography (HPLC) are also available for HPTLC.

11. Use of different development methods, such as ascending,descending, two dimensional, circular etc., is possible in HPTLCwhich is not possible in other forms of chromatography. Thisflexibility is important in method development for compounds ofsimilar polarity.

12. Since the number of samples applied on a plate is large and themobile phase used is very small, the per sample cost is low, therebyproviding an economical method of analysis.

13. The development of mass spectrometer interfaces for HighPerformance Thin Layer Chromatography - Mass Spectrometry(HPTLCeMS) is amilestone in the evolution of HPTLC, which nowhas similar capabilities to High Performance LiquidChromatography - Mass Spectrometry (HPLC-MS), in terms ofseparation and identification of samples. The use of modernspectroscopic methods such as UV, Fourier Transform Infra RedSpectroscpy (FT-IR), or Raman spectroscopy has increased thespecificity and detection power of HPTLC.

14. Recent developments in ultraperformance TLC (UPTLC)using layers 10e12 mm thick and shorter development lengths ofup to 5e6 cm has further reduced separation times and mobilephase consumption, and increased sample throughput byincreasing the number of samples that can be simultaneouslyseparated.

17.3 PHARMACEUTICAL APPLICATIONS 453

17.2 QUANTITATIVE ANALYSIS

Quantitative analysis by HPTLC is normally performed by one of twomethods.

17.2.1 Area Comparison Method

In this method, replicate spots (n¼ 3 or 6) of known concentration ofpure drug are spotted on a TLC plate along with replicate spots of thesample solution. The areas of the standard and sample spots, afterdevelopment, are calculated and averaged. The area of the standard iscompared with the area of the sample and since concentration of thestandard is known, the concentration of sample can be calculated. This isa simple and quick method.

17.2.2 Calibration Curve Method

In this method, five varied concentrations of the standard are spotted inreplicate (n¼ 3 or 6) along with the sample in replicate (n¼ 3 or 6). Afterdevelopment of the chromatogram, the average area of each concentra-tion is plotted against the concentration and a calibration curve is con-structed. The concentration of sample can be found by calculating theaverage area for the sample and with the aid of the calibration curvetransposing the area to a concentration. This method is time consumingbut more reliable.

17.3 PHARMACEUTICAL APPLICATIONS

17.3.1 Single Drug Formulation Analysis

An HPTLC method can be developed for formulations containing asingle active component by identifying a mobile phase that separatesthe active ingredient from any matrix components. Ideally an Rf valuebetween 0.3 and 0.7 is selected. The peak area for the active ingredientshould be reproducible and the peak shape sharp and without tailing.The method is then validated as per International Conference onHarmonization (ICH) guidelines. The results can be compared with theofficial method or a reported method to further confirm the accuracy ofthe developed method.

The following two examples illustrate this kind of application:

1. Analysis of metformin tablets [1] (Figure 17.1; Tables 17.1 and 17.2):


HPTLC Conditions

FIGURE 17.1 Dens

TABLE 17.1

Formulation

Bigomet

Glycomet

Biciphage

an¼ 6.

Mobile phase
Ammonium sulfate (0.5%):2-propanol:methanol (8.0:1.6:1.6 v/v/v)
Detection wavelength
238 nm
Rf
0.50� 0.03
Position of solventfront

8 cm

Saturation time
30 min
TLC plates
Merck silica gel 60 F254 on an aluminum support (20� 10 cm)
itogram of metformin hydrochloride.

Assay Results for Metformin

Label Claim (mg) Drug Content (%)a % RSD

250 100.24 0.36

500 99.82 0.98

850 101.00 1.30

TABLE 17.2 Recovery Study for Metformin

Formulation

Amount of Drug

Added (%)

Theoretical

Content (ng)

Amount of MET

Recovered (ng)a % Recovery

Glcomet 80 18,000 18,018 100.10

100 20,000 19,846 99.93

120 22,000 21995.6 99.98

Bigomet 80 9000 9003.6 100.04

100 10,000 9997 99.97

120 11,000 11031.9 100.29

Biciphage 80 30,600 30544.92 99.82

100 34,000 33874.2 99.63

120 37,400 37171.86 99.39

an¼ 6.


2. Analysis of levocetirizine hydrochloride tablets [2] (Figure 17.2;Tables 17.3 and 17.4):

HPTLC Conditions

FIGURE 17.2 Densitogram

Mobile phase
Ethyl acetate:methanol:ammonia (9:2.5:1.5 v/v/v)
230 nm
Rf
0.50� 0.02
Position of solvent front
8 cm
Saturation time
20 min
TLC plates
Merck silica gel 60 F254 on an aluminum support (20 � 10 cm)
of levocetirizine hydrochloride.

TABLE 17.3 Assay Results for Levocetirizine Hydrochloride (n¼ 6)

Okacet-L Levocetirizine (5 mg)

Levocetirizine Found (mg per Tablet)

Mean ± SD (n[ 6) Recovery (%)

First lot 4.92� 0.32 98.41

Second lot 4.93� 0.09 98.50

TABLE 17.4 Recovery Study for Levocetirizine Hydrochloride

Label Claim

(mg per Tablet)

Amount

Added (mg)

Total

Amount (mg)

Amount Recovered

(mg) ±% RSD % Recoverya

5 4 (80%) 9 9.00� 1.00 100.05

5 5 (100%) 10 9.83� 0.76 98.38

5 6 (120%) 11 11.11� 0.99 101.09

an¼ 6.


17.3.2 Two-Component Formulation Analyses

Method development for a two-component formulation requiresmore skill and expertise as two drugs of likely different polarity requireseparation with adequate resolution with a single mobile phase. Thepeak purity of the resolved drugs should be checked by overlaying thespectra of the pure drugs obtained in the same development. Furtherthe ICH guidelines need to be applied to the developed method forvalidation.

The following are two examples of this kind of application:

1. Analysis of tablets of simvastatin and ezetimibe [3] (Figure 17.3;Tables 17.5 and 17.6):

Mobile phase Toluene:2-propanol (8:2 v/v)

240 nm
Rf
0.48� 0.01 (simvastatin)
0.53� 0.01 (ezetimibe)

15 cm
Saturation time
20 min
TLC plates

TABLE 17.5 Assay Results for Simvastatin and Ezetimibe

Drug

Label Claim

(mg per Tablet)

Amount

Found (mg)

Drug

Content (%)a RSD (%)

Simvastatin 10 9.69 96.93� 0.28 0.28

Ezetimibe 10 9.74 97.73� 0.25 0.25

an¼ 3.

FIGURE 17.3 Densitogram of simvastatin and ezetimibe.

TABLE 17.6 Recovery Study for Simvastatin and Ezetimibe

Drug

Label Claima

(mg per Tablet)

Amount AddedAmount

Recovered (mg) Recovery (%)b(%) (mg)

Simvastatin 10 80 08 07.86 98.14� 0.26

100 10 09.86 98.36� 0.17

120 12 11.89 99.00� 0.26

Ezetimibe 10 80 08 07.93 99.16� 0.44

100 10 09.90 98.97� 0.28

120 12 11.87 98.85� 0.40

aFormulation: Simvas-EZ, Micro Labs, Pondicherry, India.bMean� standard deviation (n¼ 6).



2. Analysis of tablets of paracetamol and aceclofenac [4] (Figure 17.4;Table 17.7):

Mobile phase Toluene:2-propanol:25% ammonia(8:7:1 v/v/v)

FIGURE 17.4 Densitogwork carried out at Anchr

TABLE 17.7 A

Drug

Paracetamol

Aceclofenac

an¼ 6.

254 nm
Rf
0.48� 0.01 (aceclofenac)
0.67� 0.01 (paracetamol)

15 cm
Saturation time
20 min
TLC plates
ram of aceclofenac (1) and paracetamol (2). This is based on theom Test Lab Pvt. Ltd. Mumbai, India.

ssay Results for Paracetamol and Aceclofenac

Drug Content (%)a RSD (%)

98.75� 0.28 to 102.25� 0.25 0.28

98.75� 0.28 to 102.25� 0.25 0.25


17.3.3 Three-Component Formulation Analysis

Method development for a three-component formulation is morechallenging as the mobile phase should be able to adequately resolvethe three drugs, which should each have appropriate Rf values. Themethod should be validated according to ICH guidelines. Althoughmethod development may be lengthy, the per sample analysis timefor formulations is short increasing the productivity of quality controllaboratories.

1. Simultaneous analysis of atenolol, hydrochlorothiazide, andamlodipine [5] (Figure 17.5; Tables 17.8 and 17.9):

Mobile phase Chloroform:methanol:acetic acid (8:2:0.2 v/v/v)

700

600

500

400

300

200

100

0–0.11 0.00

AU

FIGURE 17.5 Densitogramamlodipine besylate (3) Rf (0.5

232 nm
Rf
0.22� 0.02 (atenolol)
0.36� 0.02 (hydrochlorothiazide)

0.55� 0.02 (amlodipine besylate)

8 cm
Saturation time
30 min
TLC plates
0.20 0.40 0.60 0.80 Rf

1

2

3

of atenolol (1) Rf (0.22), hydrochlorothiazide (2) Rf (0.36), and5).

TABLE 17.8 Assay Results for Atenolol, Hydrochlorothiazide and Amlodipine(n¼ 6)

Atenolol (50 mg)

Atenolol Found (mg per Tablet)


First lot 49.93� 6.73 99.86

Second lot 50.04� 6.60 100.08

Hydrochlorothiazide (25 mg)

Hydrochlorothiazide Found (mg per Tablet)


First lot 24.89� 3.67 99.56

Second lot 24.95� 3.55 99.80

Amlodipine Besylate (5 mg)

Amlodipine Besylate Found (mg per Tablet)


First lot 5.01� 1.21 100.20

Second lot 4.99� 1.18 99.80

TABLE 17.9 Recovery Study for Atenolol, Hydrochlorothiazide and AmlodipineBesylate (n¼ 6)

Label Claim

(mg per Tablet)

Amount

Added (mg)

Total

Amount (mg)

Amount Recovered

(mg) ±% RSD % Recovery

ATENOLOL

50 40 (80%) 90 89.92� 0.62 99.91

50 50 (100%) 100 100.21� 1.01 100.21

50 60 (120%) 110 109.66� 0.73 99.69

HYDROCHLOROTHIAZIDE

25 20 (80%) 45 44.78� 0.34 99.51

25 25 (100%) 50 50.23� 0.67 100.46

25 30 (120%) 55 54.89� 0.54 99.80

AMLODIPINE BESYLATE

5 4 (80%) 9 8.98� 0.63 99.77

5 5 (100%) 10 9.95� 0.87 99.58

5 6 (120%) 11 11.03� 0.98 100.27


2. Simultaneous analysis of paracetamol, diclofenac potassium, andfamotidine [6] (Figure 17.6; Tables 17.10 and 17.11):


Mobile phase Toluene:acetone:methanol:formic acid (5:2:2:0.01, v/v/v/v).

FIGURE 17.6 DensitogramReprinted from The Journal of A

Copyright by AOAC INTERNA

TABLE 17.10 Assay Resul

Drug

Paracetamol

Diclofenac potassium

Famotidine

an¼ 6. Two different batches of the sa

274 nm
Rf
0.17� 0.03 (famotidine)

0.75� 0.02 (diclofenac potassium)

8 cm
Saturation time
30 min
TLC plates
of famotidine (1), paracetamol (2), diclofenac potassium (3).OAC INTERNATIONAL, J. AOAC Int. 2010, 93(3), 765e771.

TIONAL.

ts for Paracetamol, Diclofenac Potassium, and Famotidine

Label Claim Amount Found, %a

325 97.5

50 96.3

20 97.1

me brand were used for analysis by the developed HPTLC method.

TABLE 17.11 Recovery Study for Paracetamol, Diclofenac Potassium, andFamotidine

Drug

Label Claim

(mg per

Tablet)

Amount

Added

(%)

Total

Amount

(mg)

Amount

Recovered

(mg) ±% RSD

%

Recoverya

Paracetamol 325 80 585 564� 1.27 96.41

100 650 638� 0.61 98.15

120 715 690� 0.45 96.50

Diclofenacpotassium

50 80 90 87.6� 0.52 97.33

100 100 96.3� 1.14 96.30

120 110 105. �0.74 95.45

Famotidine 20 80 36 34.5� 1.02 96.48

100 40 38.3� 1.32 95.75

120 44 42.1� 0.85 95.68

an¼ 6.


3. Simultaneous analysis of paracetamol, caffeine citrate, andpropyphenazone [7] (Figure 17.7; Table 17.12):

Mobile phase Dioxane:chloroform (2.5:7.5 v/v).

273 nm
Rf
0.50� 0.03 (caffeine)

0.85� 0.02 (propyphenazone)

Position of solventfront

5 cm

Saturation time
10 min
TLC plates
17.3.4 Stability Indicating Methods

The ICH guidelines recommend that every drug should be exposed tostress conditions (acid, alkali, dry heat, wet heat, oxidation and photo-stability, both sunlight and artificial light of known intensity) to assess itsinherent stability and the possiblemechanisms of its break down.Amethodwhich is capable of separating the drug in the presence of its degradedproducts is referred to as a stability indicating analytical method. The

00.00 0.50 1.00

20

80Sample

1 3

2

60

40

Rf

Det

ecto

r res

pons

e

FIGURE 17.7 Densitogram of paracetamol (1), caffeine citrate (2) and propyphenazone(3). This is based on the work carried out at Anchrom Test Lab Pvt. Ltd. Mumbai, India.

TABLE 17.12 Assay Results for Paracetamol, Caffeine Citrateand Propyphenazone

Drug Label Claim Amount Found, %a

Paracetamol 325 97.5

Caffeine citrate 50 96.3

Propyphenazone 20 97.1

aAverage of 6 readings n=6



development of stability indicating method is a challenging task since thedegradants obtained usually have similar polarities to the drug.

1. Stability indicating method for tenatoprazole [8] (Figure 17.8(a andb); Tables 17.13 and 17.14):

Mobile phase Toluene:ethyl acetate:methanol (6:4:1 v/v/v)

306 nm
Rf
0.34� 0.02
8 cm
Saturation time
30 min
TLC plates
2. Stability indicatingmethod fordesvenlafaxine [9] (Figure 17.9(a and b);Tables 17.15 and 17.16):

HPTLC Conditions

Mobile phase
Ethyl acetate:toluene:methanol:ammonia (7:2:0.5:0.5, v/v/v/v)
228 nm
Rf
0.48
Run distance
8 cm
Saturation time
30 min
TLC plates
3. Stability indicating method for tenofovir disoproxil fumarate [10](Figure 17.10(a and b); Tables 17.17 and 17.18):

HPTLC Conditions

Mobile phase
Chloroform:methanol (9:1 v/v)
260 nm
Rf
0.49� 0.03
Run distance
8 cm
Saturation time
30 min
TLC plates

900

800

700

600

500

400

300

200

100

00.1

Wavelength: 306 nm

0.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

1

Rf

AU (a)

800

700

600

500

400

300

200

100

00.1

Wavelength: 306 nm

0.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

2

3

1

Rf

AU (b)

FIGURE 17.8 (a) Densitogram of tenatoprazole. (b) Densitogram of base-treated tena-toprazole 1000 ng/spot; condition: 1 M NaOH at 80 �C for 4 h; peak 1 (tenatoprazole, Rf:0.35), peak 2 (degraded, Rf: 0.43), peak 3 (degraded, Rf: 0.59). Reprinted from The Journal ofAOAC INTERNATIONAL, J. AOAC Int. 2009, 92(2), 387e93. Copyright by AOAC

INTERNATIONAL.

TABLE 17.13 Assay Results for Tenatoprazole

Standard Added to 20 mg

Sample, mg Found, mga Recovery, % RSD, %

16 35.7 99.16 0.95

20 39.3 98.25 1.67

24 43.6 99.09 1.72

an¼ 6.


TABLE 17.14 Recovery Study for Tenatoprazole

PCP Formulationa

Tenatoprazole Foundb

Mean ± SD % Label Claim

Lot (PCP-TNZ 0307) 19.22� 1.2 96.1

Lot (PCP-TNZ 1007) 19.12� 1.4 95.6

alabel claim: 20 mg tenatoprazolebaverage of six readings n=6

600

500

400

300

200

100

0–0.18 0.02 0.22 0.42 0.02 0.82 1.02

Rf

AU

Track 1.ID: std

1

(a)

FIGURE 17.9 (a) Densitogram of desvenlafaxine. (b) Densitogram of acid degradationproduct of desvenlafaxine, condition: 1 M HCl at 80 �C for 8 h.


–0.18 0.02 0.22 0.42 0.62 0.82 1.02 Rf0

100

200

300

400

500

600AU

Track 3 .ID: 1 N HCL 8 HR

1

2

(b)

FIGURE 17.9 (Continued)

TABLE 17.15 Assay Results for Desvenlafaxine (n¼ 6)

Commercial Formulation

D-VENIZ (50 mg)

Desvenlafaxine Found (mg per Tablet)

Mean ± SD, RSD (%)

(n[ 3) Recovery (%)

First Lot 49.87� 0.38, 0.76 99.46

Second Lot 50.08� 0.60, 0.89 100.18

TABLE 17.16 Recovery Study for Desvenlafaxine (n¼ 3)

Label Claim

(mg per Tablet)

Amount

Added (mg)

Total

Amount (mg)

Amount Recovered

(mg) ± SD, RSD (%) Recovery (%)

50 40 (80%) 90 89.18� 0.74, 0.83 99.09

50 (100%) 100 99.66� 1.89, 1.90 99.66

60 (120%) 110 109.04� 1.19, 1.09 99.12


400

350

300

250

200

150

100

50

0–0.12 0.08 0.28 0.48 0.68 0.88 Rf

AU

Track 3.ID:

1

(a)

400

300

350

250

200

100

50

0

150

–0.12 0.08 0.28 0.48 0.68 0.88 Rf

AU

Track 5.ID:

1

2 3

(b)

FIGURE 17.10 (a) Densitogram of tenofovir disoproxil fumarate (30 mg/mL). Mobilephase: chloroform: methanol (9.0: 1.0 v/v); detection wavelength: 260 nm; Rf: 0.49� 0.03; po-sition of solvent front: 8 cm; saturation time: 30 min; TLC plates: Merck silica gel 60 F254aluminum (20� 10 cm). (b) Densitogram of photo degraded tenoforvir disoproxil fumarate(150 ng/spot) peak 1: tenoforvir disoproxil fumarate, peak 2 and 3 degradants.

TABLE 17.17 Assay Results for Tenofovir Disoproxil Fumarate and Comparisonwith HPLC

Label Claim (mg) Sample

HPLC HPTLC

Drug Content (%) % RSD Drug Content (%) % RSD

300 Tentide 99.69 0.002 99.47 0.05

300 Tavin 99.80 0.006 99.91 0.009


TABLE 17.18 Recovery Study for Tenofovir Disoproxil Fumarate

Label Claim

(mg)

Amount of

Drug Added (%)

Total Amount of

Drug Present (mg)

Amount

Found (mg) % Recoverya

TENTIDE

300 80 540 536.65 99.38

100 600 597.06 99.51

120 660 655.05 99.25

TAVIN

300 80 540 536.27 99.31

100 600 599.04 99.84

120 660 654.12 99.11

an¼ 3.


17.3.5 Determination of Content Uniformity

All national pharmacopoeia require that each dosage form is testedfor content uniformity. For tablets, as an example, 10e20 tablets aretaken and assayed individually. The batch passes or fails the testdepending upon how many tablets fall outside the specification. Thenumber of tablets which must pass the test is given by the pharmaco-poeia depending upon the amount of active ingredient or averageweight of the tablets.

Thus all other methods of analysis would require the repetition of thesame experiment 10 or 20 times, which is very tedious and timeconsuming. Using HPTLC, once the individual tablet solutions are ready,all solutions can be applied to a single plate and separated simulta-neously, saving time and reducing the release time of the batch.

The prerequisite is the development of a validated HPTLC method forthe drug. Following two examples will illustrate this application:

1. Determination of content uniformity of alprazolam tablets [11](Figures 17.11 and 17.12; Table 17.19):

HPTLC Conditions

Mobile phase
Chloroform:ethanol (9:1 v/v)
225 nm
Rf
0.40
Run distance
7 cm
Saturation time
20 min
TLC plates

0.0

50.0

100.0

150.0

200.0

250.0

a

Track no.

bc

300.0

(AU)

400.0D

etec

tor r

espo

nse

3D plot of 3 tracksDensitogram of alprazolam standard & sample,standard (90%): track-a; pooled sample: track-b; standard (110%): track-c

FIGURE 17.11 Densitogram of alprazolam standard and sample. This is based on thework carried out at Anchrom Test Lab Pvt. Ltd. Mumbai, India.

0.0

50.0

100.0

150.0

200.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17250.0

300.0

(AU)

400.0

Det

ecto

r res

pons

e

3D plot of all tracks

Densitogram of all tracks

Track no.

Densitogram of alprazolam standards(light) and the samples (dark) For detailed experimental and chromatographic conditions, refer to protocol no: 1 Tracks identification: Standard (100%) :3,8,10,13,15, Standard (85%): 5 and Standard (115%): 7, samples:1,2,4,6,9,11,12,14,16,17

FIGURE 17.12 Densitogram of alprazolam tablets. This is based on the work carried outat Anchrom Test Lab Pvt. Ltd. Mumbai, India.


TABLE 17.19 Assay Results for Alprazolam Tablets

Assay %a Minimum Found Maximum Found

Commercial formulationalprazolam (1 mg)

Mean¼ 99.34RSD (%)¼ 1.28

96.76 101.76

an¼ 5.


2. Determination of content uniformity of hydrochlorothiazide andlisinopril tablets [12] (Figures 17.13 and 17.14; Table 17.20):

HPTLC Conditions

0–0.200.0

100.0

200.0

300.0

400.0

500.0

(AU) Sta

700.0

Det

ecto

r res

pons

e

FIGURE 17.13 Densitois based on the work carrie

Mobile phase
n-Butanol:methanol:water (3:1:1 v/v/v)
200 nm
Rf
Lisinopril 0.12
Hydrochlorothiazide 0.73

Run distance
7 cm
Saturation time
20 min
TLC plates
.00 0.20 0.40 0.60 0.80 (Rf) 1.20

ndard

1

2

Rf

gram of lisinopril (1) and hydrochlorothiazide (2) standard. Thisd out at Anchrom Test Lab Pvt. Ltd. Mumbai, India.

0.00 0.20 0.40 0.60 0.80 (Rf) 1.20–0.200.0

100.0

200.0

300.0

400.0

500.0

(AU) Sample

1

2

700.0

Rf

Det

ecto

r res

pons

e

FIGURE 17.14 Densitogram of lisinopril (1) and hydrochlorothiazide (2) tablets. This isbased on the work carried out at Anchrom Test Lab Pvt. Ltd. Mumbai, India.

TABLE 17.20 Assay Results for Hydrochlorothiazide and Lisinopril Tablets

Name of the Drug Assay (%)a Minimum Found Maximum Found

Lisinopril(label claim 5 mg)

Mean¼ 98.98RSD (%)¼ 1.81

96.36 101.30

Hydrochlorothiazide(label claim 12.5 mg)

Mean¼ 97.53RSD (%)¼ 4.44

86.64 101.07

an¼ 5.


17.3.6 Bulk Drug Purity Profiles

Bulk drugs contain impurities even if they pass meet the criteria set out innational pharmacopeias. Consequently, impurity profiling is a major interestin pharmaceutical analysis. HPTLC has been used successfully for this pur-pose. A high concentration of sample is applied to the layer, which is devel-oped in amobile phase capable of providing adequate resolution between the


pure drug and the impurities present. The impurities can be isolated bypreparative TLC and the structure of the impurities determined by conven-tional spectroscopic methods, such as Nuclear Magnetic Resonance(NMR),Mass Spectrometry(MS), Infra Red Spectroscopy (IR) and Ultraviolet Spec-troscopy(UV). TLCeMS is increasingly being used for this application.

The following example of famotidine illustrates this application [13](Figure 17.15 (a) and (b)):

(a) (b)

FIGURE 17.15 (a) Densitogram of impurity profiling of Famotidine: 1,2 and 4 impu-rities;3 famotidine (b) 3-D plot of densitometric scans of above samples at UV 254 nm.This work has been carried out at Anchrom Test Lab Pvt. Ltd. Mumbai, India

17.3.7 Standardization of a Herbal Drug Formulation

TLC has been used for decades for identification of different constit-uents of herbal drugs. However, the ability of HPTLC to perform quan-titative analysis is particularly helpful for both identification (e.g.,authentication) and quantitation to standardize herbal formulations. Dueto their complexity herbal formulations are difficult to standardize byother approaches.

Typically, a prominent biomarker/chemical marker is identified in theauthentic sample of the crude drug and an HPTLC method is developedfor its determination. Once a validated method is available, then either acrude drug sample can be authenticated or a herbal formulation can bestandardized by determining the presence of the maker qualitatively andquantitatively.


1. Example: Rapid densitometric method for estimation of alizarin andbetulinic acid in a polyherbal formulation [14] (Figures 17.16 and17.17(a and b); Table 17.21):

HPTLC Conditions

FIGURE 17.16 (a) Dedensitometric scans of aboAnchrom Test Lab Pvt. Ltd

Mobile phase
Toluene:ethyl acetate:formic acid (9:1.5:0.5 v/v/v)
287 nm
Rf
Alizarin �0.58� 0.02
Betulinic acid �0.52� 0.02

Run distance
8 cm
Saturation time
30 min
TLC plates
nsitogram of impurity profiling of Famotidine; (b) 3-D plot ofve samples at UV 254 nm. This work has been carried out at. Mumbai, India.

2. Development and validation of an HPTLC method for thesimultaneous estimation of corilagin, gallic acid, and ellagic acid in apolyherbal formulation [15] (Figures 17.18 and 17.19; Table 17.22):

FIGURE 17.17 (a) Densitogram of crude extract of Syzygium jambolanum containingAliz-arin and Betulinic acid. (b) Densitogram of formulation containing alizarin and betulinicacid.

TABLE 17.21 Assay Results for Formulations Containing Alizarinand Betulinic Acid (n¼ 3)

Samples

Drug Content (%w/w± SD)

Alizarina Betulinic Acida

Crude extract 1.77� 0.120 1.58� 0.166

Marketed formulation 0.40� 0.022 0.39� 0.012

aValues for Alizarin and Betulinic acid are positive only the sign of þ and � together is for

indicating Standard Deviation



HPTLC Conditions

350

300

250

200

150

100

50

0–0.10 0.10 0.30 0.50 0.70

AU

1

FIGURE 17.18 Densitoacid (Rf 0.63) at 600 ng spo

300

350

250

200

150

100

50

0 –0.10 0.1

AU

FIGURE 17.19 Densitocorilagin (Rf 0.45), gallic ac

Mobile phase
n-Butanol:water methanol:formic acid (6:1:0.1:0.8 v/v/v/v)
283 nm
Rf
Corilagin �0.44
Ellagic acid �0.63

Gallic acid �0.73

Run distance
8 cm
Saturation time
30 min
TLC plates
350

300

250

200

150

100

50

0

350

300

250

200

150

100

50

00.90 Rf –0.10 0.10 0.30 0.50 0.70 0.90 Rf –0.10 0.10 0.30 0.50 0.70 0.90 Rf

AU AU

1

1

gram of pure corilagin (Rf 0.44), gallic acid (Rf 0.79), and ellagict (1).

0 0.30 0.50 0.70 0.90 Rf

1 2

3

4

gram of polyherbal formulation of Phyllanthus amarus containingid (Rf 0.80), and ellagic acid (Rf 0.65).

TABLE 17.22 Assay Results for the Poly Herbal Formulation ContainingCorilagin, Gallic Acid, and Ellagic Acid

Samples Corilagina Gallic Acida Ellagic Acida

Commercial crude powderMarketed formulation

0.030% 0.035% 0.072%

Formulation 1 0.355% 0.373% 0.121%

Formulation 2 0.013% 0.080% 0.104%

aMean� standard deviation (n¼ 3).

REFERENCES 477

17.4 CONCLUSIONS

HPTLC is being increasingly used for applications in pharmaceuticalanalysis. Among its advantages are rapid analysis, simpler samplepreparation, reduced cleanup requirements, and most important of all,simultaneous analysis of several samples and standard under identicalconditions, making system suitability test unnecessary. The availability ofdifferent stationary phases has increased the applications of HPTLC todiverse samples incompatible with separation on bare silica gel. Theapplication of HPTLC to content uniformity testing, bulk drug impurityprofiling, and polyherbal formulations demonstrates how modern TLChas succeeded in expanding its scope to more complex sample types. Theability of HPTLC to separate a large number of samples simultaneously isunique and contributes to decreased per sample analysis costs. The futurewill definitely see more application of HPTLC in pharmaceutical analysisand increasing adoption of this method by national pharmacopoeias.

References

[1] Havele S, Dhaneshwar S. Estimation of metformin in bulk drug and formulation byHPTLC. J NanomedNanotechnol 2010;1:102. http://dx.doi.org/10.4172-7439, 1000102.

[2] Bhusari VK, Dhaneshwar SR. Application of stability indicating TLC method forthe quantitative determination of Levocetirizine in pharmaceutical dosage forms.Intern J Adv Pharm Sci 2010;1:387e94. http://dx.doi.org/10.5138/ijaps,(2010),0976.1055.01048.

[3] Dhaneshwar SS, Deshpande P, Patil M, Vadnerkar G, Dhaneshwar SR. Developmentand Validation of a method for Simultaneous Densitometric Analysis of Simvastatinand Ezetimibe as the bulk drug and in the tablet dosage form. Acta Chromatogr2008;20:71e9. http://dx.doi.org/10.1556/A.Chrom.20.2008.1.6.

[4] Sethi PD. HPTLC high performance thin layer chromatography-quantitative analysis ofpharmaceutical formulations, vol. 2. NewDelhi: CBS Publishers and Distributors; 2013.p. 518e19.

[5] Bhusari VK, Dhaneshwar SR. Validated HPTLCmethod for simultaneous estimation ofAtenolol, Hydrochlorothiazide and Amlodipine Besylate in bulk drug andformulation. Intern J Anal Bioanal Chem 2011;1(3):70e6.

http://dx.doi.org/10.4172-7439

http://dx.doi.org/10.5138/ijaps,(2010), 0976.1055.01048

http://dx.doi.org/10.5138/ijaps,(2010), 0976.1055.01048

http://dx.doi.org/10.1556/A.Chrom.20.2008.1.6

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[6] Khatal LD, Kamble AY, Mahadik MV, Dhaneshwar SR. Validated HPTLC method forsimultaneous quantitation of Paracetamol, Diclofenac Potassium and Famotidine intablet formulation. JAOAC Intern 2010;93(3):765e71.

[7] Sethi PD. HPTLC high performance thin layer chromatography quantitative analysis ofpharmaceutical formulations, vol. 2. NewDelhi: CBS Publishers and Distributors; 2013.p. 620e21.

[8] Dhaneshwar SR, Bhusari VK, Mahadik MV. Application of a stability indicating ThinLayer Chromatographic method to determination of Tenatoprazole in pharmaceuticaldosage forms. JAOAC Intern 2009;92(2):387e93.

[9] Pawar SM, Dhaneshwar SR. Application of a stability indicating Thin Layer Chromato-graphic method for quantitation of Desvenlafaxine in pharmaceutical dosage forms. JLiq Chromatogr Rel. Technol 2012;35:499e510.

[10] Havele S, Dhaneshwar SR. Stress studies of Tenofovir Disoproxil Fumarate by HPTLCin bulk drug and formulation. Sci World J; 2012:1e6.

[11] Sethi PD. HPTLC high performance thin layer chromatography content uniformity ofpharmaceutical formulations. New Delhi: Kongposh Publications Pvt. Ltd.; 2011.p. 1e2.

[12] Sethi PD. HPTLC high performance thin layer chromatography content uniformity ofpharmaceutical formulations. New Delhi: Kongposh Publications Pvt. Ltd.; 2011.p. 98e101.

[13] Anchrom Test Lab Pvt. Ltd. Mumbai, India.[14] Kulkarni PV, Sathiyanarayanan L, Nikam AR, Mahadik KR. (M.Pharm. thesis) submit-

ted to Bharati Vidyapeeth Deemed University, Pune India; 2011.[15] Kakade T, Sathiyanarayanan L, Mahadik KR. M.Pharm. thesis submitted to Bharati

Vidyapeeth Deemed University, Pune India; 2011.




























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Instrumental Thin-Layer Chromatography || Pharmaceutical Applications of High Performance Thin Layer Chromatography