Quality Control of MAPs and their Extracted Products by HPLC and HPTLC

Karan Vasisht
M. Pharm., Ph.D. Professor of Pharmacognosy

UNIVERSITY INSTITUTE OF PHARMACEUTICAL SCIENCES PANJAB UNIVERSITY, CHANDIGARH

QC through HPLC & HPTLC

Parameters of standardization Analysis technique and validation procedure Introductory remarks on technique of HPTLC and HPLC Sources of error in HPTLC & HPLC Examples of application of techniques

Quality control of MAPs & their products

Quality control of raw material In process quality control Quality control of finished products

Quality control of raw material

APIs, phytopharmaceuticals & phytoproducts Factors known to influence quality of raw material
Climatic: temp, rainfall, day light, altitude Nutritional: soil factors, pH, nutrients & CEC Collection: part, season, age, collection time Post-harvest processes: respiration in live cells,

enzyme activity until their deactivation, decompartation of chemicals during cutting and crushing, oxidation from air & light, radiation effect, physical loss of components

Factors affecting quality during storage

Quality control of raw material

(WHO guidelines)
Identification Moisture content Foreign matter Macro & microcharacters TLC Extractives Ash value Bitter value Foaming index Haemolytic index Swelling factor Tanning test Volatile matter Microbial load Toxic residues Pesticide residue Radioactive contamination

Quality control of raw material

Methods to determine intrinsic potency Chemical content of medicinal plant material Biological testing Chemical analysis

Chemical standardization
Identification of analyte (marker) Estimation of marker in the drug material

Identification of marker
Constituents of a medicinal plant material which are chemically defined and of interest for control purposes
Chemically characterized Preferably biologically active Quantitative correlation to said activity Specific to plant
ubiquitous plant constituents to be avoided

Not amenable to maneuverability

Identification of a marker

Knowledge of active (s) Phytochemistry to be known Generated in-house or commercially procured

Procedures for marker analysis

Any analytical technique: Simple & fast Specific Inexpensive Robust with least critical variables
UV, HPLC, QTLC, IR, PS

Validation of analytical procedure

Procedure Instrument

Validation of analysis procedure (ICH guidelines)

Specificity Linearity Range Accuracy Precision Detection limit Quantitation limit Robustness

Specificity
Should specifically measure analyte of interest It is not always possible to demonstrate specificity of analysis procedure for the analyte (complete discrimination) Two or more assay procedures are necessary to demonstrate necessary level of discrimination Deduced from positive results with mixture containing analyte and negative results from mixture containing similar compounds but no analyte Especially valuable when analyzing analyte among similar compound in the mixture

Linearity & Range

Linearity evaluated across the range using dilutions of the stock solution and / or separate weighings Range should be determined by separate weighings Visually seen from the plot of signals as a function of analyte concentration Data from regression line itself may be helpful to provide mathematical estimates of degree of linearity Correlation coefficient, y-intercept, slope, regression line, degree of deviation of data points from the line are helpful in checking linearity Specified range normally defined from linearity studies

Linearity
EGCG (ng) Mean AUCSD
800

Standard plot of EGCG (HPLC)

9.85 19.70 39.35 78.50 157.50 283.20

23471 19.0
600 AUC (x103)

46026 855 88815 36 175341 2502 355954 6708 635194 3077

400 200 0 0 50 100 150 200 250 300

R =1 y = 2240.7x + 1175.4

EGCG (ng)

Accuracy
How close is the measurement to actual value Should be specified across the range of analytical procedure Application of the procedure to substance of known purity Application of the procedure to test sample after spiking Inferred from 9 measurements (triplicates of three concentrations in the range)

Precision
Repeatability from 9 measurements (triplicates of three concentration in the range) or 6 measurements at 100% of test concentration (intra-lab, same analyst, same equipment) Intermediate precision using different days, analysts and equipment Reproducibility by means of inter-laboratory trials (when procedure is recommended for wider use say pharmacopoeial procedure)

Detection limit
Several approaches Based on signal to noise ratio (> 2:1 or 3:1) Standard deviation and response (Detection limit = 3.3 SD/slope) Based on standard deviation of blank measurements Standard deviation of regression lines

Quantitation limit
Established from experiments, minimum level of analyte at which it can be reliably quantified Based on signal to noise ratio (quantitation limit = 10 standard deviation / slope)

Robustness
Extraction time Stability of analytical solutions Pertaining to HPLC:
Columns, different lots or manufacturers, pH and composition of mobile phase, temperature influence, flow rate

Accessibility

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TLC or planar chromatography

Extension of column chromatography open column chromatography Most accepted and extensively used chromatographic technique Simple, cost effective, versatile, available in all laboratories around the globe Based upon technique of stationary phase (adsorbent) and mobile phase (eluting solvent)

Steps involved in quantitative TLC

Selection of chromatographic layer Selection of mobile phase Sample application Development of plates Drying of plate; derivatization if required Chromatogram evaluation

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Selection of chromatographic layer

80% of analyses use silica gel GF plates Other options are aluminium oxide, cellulose, reverse phase (RP2, RP8 and RP18), hybrid & derivatized plates Nature of compounds define the choice Stronger adsorbent (aluminium oxide) for weakly adsorbed compounds and weak adsorbent (cellulose) for strongly adsorbed compounds Nonpolar compounds elute first on normal phase Polar components elute first on reverse phase

Selection of mobile phase

Infinite combinations and wide choice Trial and error method from existing knowledge Literature support & own experience 1 to 3-component mobile phase to be preferred over multicomponent Fresh constitution of mobile phase for each run Mix outside and add to developing chambers Chamber saturation as desired: slow, quick with filter paper, with vapours of another solvent possible in twin troughs

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Sample application
Usual concentration of applied samples 0.1 to 1 g / L for qualitative analysis and quantity may vary in quantitation based on UV absorption 1 to 5 L for spot and 10 L for band application Better results with band application in quantitation as narrow band is better suited to optics of the TLC scanner Manual, semi-auto or auto application Manual with calibrated capillaries Semi and auto-application through applicators Applicators use spray on or touch and deliver technique for application

Sample application

Band application

Spot application

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Development of plates
Best achieved in special purpose TLC chambers Improvised use of all types of chambers possible Twin-trough chambers allow saturation of chamber with different mobile phase Pre-saturation decreases Rf values and corrects side distortion of solvent front

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Drying and derivatization

Drying of plate to completely remove the mobile phase Vacuum drying in desiccator preferred Derivatization for UV insensitive compounds Dipping or spraying with chromogenic reagents Controlled dipping preferred over spraying and dip chambers available

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Chromatogram evaluation
Scanning of tracks in TLC scanner in transmittance, reflected or fluorescence mode Multi-wavelength scanning possible UV scanning standard on all scanners Transmittance and fluorescence modes optional Data acquisition through standard softwares provided with the instrument

Analysis and interpretation of data

Analysis of data like other analytical tools Validation of procedure Single or double point calibration or from standard plot

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HPTLC vs TLC
Smaller size of adsorbent particles (100 vs 250 m) Narrow range of particle size, more homogenous layers Better resolution over shorter runs More precision and accuracy Normal run 3-5 cm vs 8-10 cm Because of shorter run analysis time is reduced But fairly expensive in comparison to TLC Not necessarily essential in all analyses Used where resolution on ordinary plates is poor

Introducing precision in TLC

Use of HPTLC plates Automatic spray-on sample application technique Use of TLC chambers Selecting optimal range for analysis Correct instrument parameters: slit dimensions, measuring wavelength, scanning speed Base line correction to maximize the signal to noise ratio Derivatization introduces more error; homogenous application of reagent minimizes the scope of error

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TLC finger print profile

Sample : Methanolic extract STD : DPH-1 Solvent system: Touene: ethyl acetate (9.5:0.5) Spray reagent: Anisaldehyde - sulphuric acid Visualization: 110 0C for 10 min or UV short wavelength without spray
Under UV short wavelength without spray After anisaldehyde derivatization

Quantitative estimation of DPH-1

Test solution: 5 g powdered whole plant soxhlet for 4 h in methanol Dilution: 5g / 50 ml x 1/10 ml with methanol Std solution: DPH-1 Dilution: 4.95 mg / 10 x 0.5/10
AUC 5000 4000 3000 2000 1000 0 0 20 40 Amount of DPH-1 in ng 60 80 y = 53.673x + 463.13 R = 0.9948
2

Calibration curve of DPH-1

Calibration curve: Six different concentration from 0.5 to 3.0 L of std Solvent system: Toluene: Ethyl acetate (9:1) Instrument: Camag TLC scanner Wave length: 305 nm Estimation of DPH-1: AUC of DPH in 1 L spotted test solution and estimation using calibration curve Result ( % of DPH-1): 0.26 % in sample 1 and 1.45 % in sample 2

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Saussurea costus
Traditional uses
Anti-inflammatory Antiasthmatic

Chemical constituents
Costunolide Dehydrocostus lactone Alantolactone Isoalantolactone -Cyclocostunolide Isodehydrocostus lactone Isozaluzanin C

TLC fingerprint profile of Saussurea costus

Solvent system: Toluene : Ethyl acetate (95 : 5) Plates: Silica gel 60 F254 ; 0.2mm thick Spray reagent: Anisaldehyde-sulphuric acid

1. Costunolide 2. Pet. ether extract

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Isolation of costunolide and dehydrocostus lactone

Recrystallization Pet Ether : EtOAc
O O

Dehydrocostus lactone

CC Silica gel
Freezer crystallization

Drug

Pet Ether Maceration

Extract

Costunolide

M.Liquor

Costunolide

Crystallization

Standard curve of costunolide and dehydrocostus lactone

Stock solutions: 10.5 & 11.1 mg/dL in methanol Sample volume: 10 L Solvent system: Tol : Et Ac :: 97 : 3 TLC Plates: Silica gel 60F254 0.2mm Scanning : 220 nm Linearity range: 100 to 600 ng Result CT 0.46 - 1.00 DCT 0.81 - 1.21
CT (ng) 105 210 315 420 525 630 DCT (ng) 111 222 333 444 555 666 Mean AUCSD 836 15 1618 69 2326 10 3022 71 3513 64 4070 44 Mean AUCSD 520 48 1069 24
AUC
AUC 5000 4000 3000 2000 1000 0 0 200 400 Conc ng 600 800

Costunolide Standard Curve

y = 6.1363x + 309.07 R2 = 0.993

Dehydrocostuslactone calibration
3500 3000 2500 2000 1500 1000 500 0 0 200 400 Conc ng y = 4.2224x + 92.933 R2 = 0.9988

1511 29 1990 5 2409 18 2901 13

600

800

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TLC analysis of costunolide and dehydrocostus lactone

TLC Chromatogram at wavelength 220nm A. Costunolide B. Test solution

Analysis steps Weigh exactly about 5 g root powder Extract in Soxhlet with 50 ml methanol for 4h Filter and make up the volume to 50 mL Take 1 ml, dilute to 50 mL and use 10 L Develop the plate and scan at 220 nm Record the AUC and calculate the concentration from the standard curves

TLC multiwavelength scanning and spectra recording

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HIGH PERFORMANCE (PRESSURE) LIQUID CHROMATOGRAPHY

Mechanism Partition chromatography (distribution between liquid - liquid phase) Adsorption chromatography Type Closed column chromatography The mobile phase (a liquid) is pumped at moderately high pressures through a narrow-bore column. The stationary phase consists of solid particles of very small size and large surface area or high boiling point liquid chemically bonded to surface of adsorbent

HPLC instrumentation Pumps Injector Column Detector Data processing

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HPLC instrumentation

Instrumentation

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HPLC
Solvent reservoir:
glass or stainless steel

Sonication / helium gas purging for degassing: Pumps:

solvents must be degassed to eliminate formation of bubbles and base line noise are to be primed to remove any bubble that may be in solvent delivery line normally a linear ramp is used to change the composition but convex and concave ramping curves are also available fixed volume loop or variable volume injection normal phase or reverse phase, several sizes, packing material, support material UV, PDA, RI, EC, FL, ELSD, CD Several good softwares from manufacturers or from third parties

Gradient controller: Injection port: Column:

Detector:

Data module / Programmer:

Factors affecting HPLC analysis

Column temperature ( 0.2 C)
sample solubility, solute diffusion, mobile phase viscosity, column plate number

Sources of error in gradient elution

Mobile phase composition, flow-rate, strength

Checking system suitability and pump and injector precision

Repeated injections (6), constancy of Rt of last eluted peak for long term flow accuracy; average value of peak area and its SD for short term flow accuracy

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Factors affecting retention time

Mobile phase composition
Capacity factor varies exponentially with mobile phase therefore very large effect on Rt. For 1% precision control composition of mobile phase within 0.1%

Flow rate
Normal pump variations 0.3% same magnitude of variation in Rt

Column temperature
At 25 C variation of 3 C will transform to 1.5% variation in the Rt. Control to about 0.4 C for 1% and 0.04 C for high precision of 0.1%.

Recording errors
Variation of 0.38 s at Rt 6.283 min (RSD 0.1%), 0.2 s at 8.119 (RSD 0.04%)

Peak areas
Area of peak = Detector response x wt of solute / flow rate Flow rate variation will elicit same variation in peak area; reduction of 1% will increase the peak area by 1% and height by 0.3% Changes in temperature will introduce error in peak area as it does for retention time Same is true for composition change The performance of the integration system will contribute to variation in measured peak areas Peak start and end measurements Inversely proportional to signal to noise ration, low noise better precision

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Extra-column effects
Injection Volume Injector Connection Tubing Endfittings and Frits Detector Volume

Injection volume

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Incorrect sample solvent

Connecting tubes

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An example: HPLC ANALYSIS OF EGCG IN TEA

Standard curve
Stock solution: 0.0984 mg/ml methanol Injection volume: 5 L Linearity range: 10 to 280 ng
EGCG (ng) Mean AUCSD
800

Standard plot of EGCG (HPLC)

9.85 19.70 39.35 78.50 157.50 283.20

23471 19.0
AUC (x103)

46026 855 88815 36 175341 2502 355954 6708 635194 3077

600 400 200 0 0 50 100 150 200 250 300

R =1 y = 2240.7x + 1175.4

EGCG (ng)

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Effect of solvent on EGCG extraction

70 60

EGCG (mg/g)

50 40
48.39 53.6

30 20 10 0

57.12

Methanol

Water 60

Water 80

Ethyl acetate

Water extraction: Effect of temperature

60 50
EGCG (mg/g)

40 16.44 30 20 10 0

40

60

40.65

80
Temperature

42.72

98

53.6

4.32

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Methanol extraction: temperature effect

60 50

EGCG (mg/g)

59.06

40 30 20 10 0

40

29.98

60
Temperature

Water extraction: time influence

53.62 56 54 EGCG (mg/g) 48.48 52 50 48 46 44 42 5 10 15 30 45 60 75 90 Time (min) 54.3 53.6

49.55

48.68

80

49.45

48

49.15

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Sample : solute ratio

EGCG (mg/g)
80 60 40 20 0 10 :1 1 :25 1 :50 1 00 :1 1 50 :1 1 :200 1 :400

Sample : solvent

Effect of particle size

60 58 EGCG (mg/g) 56 54

56.54

58.85

52 50 48

Coarse

55.88

Moderately Coarse

Moderately Fine

Fine

56.34

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HPLC analysis details

Reverse phase C18, -bondapak, water: methanol: acetic acid (70: 30: 0.5), 1 ml/min, 280 nm Waters automated gradient controller, 510 pumps, U6K injector, 481 detector & 746 data module Analysis temperature range: 25C 2

Test chromatogram and result

80 70 60 EGCG (mg/g) 50
68.89 64.38

40 30 20 10

68.35

58.52

57.82

51.47

50.18

45.44

44.18

42.31

38.12

PA SI -2

PA SI -9

PA SI -8

TV -2

TV -1

wa l

As h

ng ra

TR

R6

Ja

TR

Ka ng ra

Ka

hi n

Va ri e

I-2

I-2

TV -9

02 6

01 7

02 5

ty

33.91

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TLC vs HPLC analysis

Sample and mobile phase preparation do not require elaborated steps of purification, degassing, filtration etc Several samples (upto 18) can be accommodated on 20 x 20 cm plate Simultaneous analysis of test and standard under similar conditions Several analysts can work simultaneously Unlimited choice of solvent systems Enormous flexibility of derivatization with chromogenic spray reagent Multiple evaluation of developed chromatogram possible Lower analysis time in comparison to other techniques No interference from previous analysis Compound sensitivity to degradation with light and air, which is not so with HPLC

Conclusion
HPLC is the workhorse in analytical work and most rapidly advancing with highest level of sensitivity and greatest resolutions achieved not possible with HPTLC Higher sensitivity and reproducibility with HPLC but at higher cost HPTLC is the fastest chromatographic technique which is a time machine and allows to do many things in short spans The multi-component matrix of plant drugs more suited to HPTLC analysis HPLC and HPTLC allow quantification of one or more components Monitoring of changes in multicompound systems possible Both HPTLC and HPLC are indispensable in QC of MAPs

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