instrumental thin-layer chromatography || pharmaceutical applications of high performance thin...
TRANSCRIPT
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of high performance thin layer chromatography(HPTLC) in pharmaceutical analysis represents an important milestonehaving superseded conventional TLC where it has several distinctadvantages.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.
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):
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE454
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 nmRf
0.50� 0.03Position of solventfront
8 cm
Saturation time
30 minTLC 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.
17.3 PHARMACEUTICAL APPLICATIONS 455
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)Detection wavelength
230 nmRf
0.50� 0.02Position of solvent front
8 cmSaturation time
20 minTLC 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. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE456
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)
Detection wavelength
240 nmRf
0.48� 0.01 (simvastatin)0.53� 0.01 (ezetimibe)
Position of solvent front
15 cmSaturation time
20 minTLC plates
Merck silica gel 60 F254 on an aluminum support (20� 10 cm)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).
17.3 PHARMACEUTICAL APPLICATIONS 457
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE458
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.
Detection wavelength
254 nmRf
0.48� 0.01 (aceclofenac)0.67� 0.01 (paracetamol)
Position of solvent front
15 cmSaturation time
20 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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 PHARMACEUTICAL APPLICATIONS 459
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
Detection wavelength
232 nmRf
0.22� 0.02 (atenolol)0.36� 0.02 (hydrochlorothiazide)
0.55� 0.02 (amlodipine besylate)
Position of solvent front
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (20� 10 cm)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)
Mean ± SD (n[ 6) Recovery (%)
First lot 49.93� 6.73 99.86
Second lot 50.04� 6.60 100.08
Hydrochlorothiazide (25 mg)
Hydrochlorothiazide Found (mg per Tablet)
Mean ± SD (n[ 6) Recovery (%)
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)
Mean ± SD (n[ 6) Recovery (%)
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
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE460
2. Simultaneous analysis of paracetamol, diclofenac potassium, andfamotidine [6] (Figure 17.6; Tables 17.10 and 17.11):
17.3 PHARMACEUTICAL APPLICATIONS 461
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
Detection wavelength
274 nmRf
0.17� 0.03 (famotidine)0.62� 0.03 (paracetamol)
0.75� 0.02 (diclofenac potassium)
Position of solvent front
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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.
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE462
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).
Detection wavelength
273 nmRf
0.25� 0.03 (paracetamol)0.50� 0.03 (caffeine)
0.85� 0.02 (propyphenazone)
Position of solventfront
5 cm
Saturation time
10 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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
17.3 PHARMACEUTICAL APPLICATIONS 463
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE464
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)
Detection wavelength
306 nmRf
0.34� 0.02Position of solvent front
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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)Detection wavelength
228 nmRf
0.48Run distance
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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)Detection wavelength
260 nmRf
0.49� 0.03Run distance
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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.
17.3 PHARMACEUTICAL APPLICATIONS 465
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.
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE466
–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
17.3 PHARMACEUTICAL APPLICATIONS 467
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
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE468
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 PHARMACEUTICAL APPLICATIONS 469
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)Detection wavelength
225 nmRf
0.40Run distance
7 cmSaturation time
20 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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.
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE470
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.
17.3 PHARMACEUTICAL APPLICATIONS 471
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)Detection wavelength
200 nmRf
Lisinopril 0.12Hydrochlorothiazide 0.73
Run distance
7 cmSaturation time
20 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm).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. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE472
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
17.3 PHARMACEUTICAL APPLICATIONS 473
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.
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE474
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)Detection wavelength
287 nmRf
Alizarin �0.58� 0.02Betulinic acid �0.52� 0.02
Run distance
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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
17.3 PHARMACEUTICAL APPLICATIONS 475
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE476
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)Detection wavelength
283 nmRf
Corilagin �0.44Ellagic acid �0.63
Gallic acid �0.73
Run distance
8 cmSaturation time
30 minTLC plates
Merck silica gel 60 F254 on an aluminum support (10� 10 cm)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
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[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.
17. PHARMACEUTICAL APPLICATIONS OF HIGH PERFORMANCE478
[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.