QUALIFICATION OF

ANALYTICAL EQUIPMENTS:
HPTLC and LC- MASS SPECTROMETRY

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PRINCIPLE
• HPTLC have similar approach and employ the
same physical principles of TLC (absorption
chromatography) i.e. the principle of separation is
absorption .
• The mobile phase solvent flows through because of
capillary action . The components move according
to their affinities towards the absorbent . The
component with more affinity towards the
stationary phase travels slower . The component
with lesser affinity towards the stationary phase
travels faster . Thus the components are separated
on a chromatographic plate .
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 SALIENT FEATURES OF HPTLC
• It is simple to learn and operate.
• Accuracy and precision of quantification is high.
• Samples rarely require cleanup. Low maintenance cost.

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STEPS INVOLVED IN HPTLC
DEVELOPMENT
• Selection of chromatographic layer
• Layer pre-washing of precoated plates
• Layer Pre-conditioning/Activation of precoated plates
• Selection and Optimization of mobile phase
• Sample and standard preparation
• Application of sample and standard
• Chromatographic development
• Detection of spots
• Scanning and documentation of chromo plate.

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INSTRUMENTATION
• Sample applicator
• Developing chamber
• Derivatization device
• Immersion device
• Plate heater
• Scanning densitometer
• Other accessories like:
(i) Plate coater
(ii) Drying rack
(iii) Plate cutter.
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A. SAMPLE APPLICATOR
• The samples are applied onto the layer as spots or bands.
• The samples are applied onto the layer as spots or bands.
• Usual concentration of applied samples 0.1 to 1 ȝJ / ȝO for
qualitative Analysis and quantity may vary in quantitation based
on UV absorption 1 to 5 ȝO for spot and 10 ȝ/ for band
application.
• Precision of the applied volume, exact positioning and
compactness of the application zones are important for the quality
of the analysis.
• Three types of sample applicators are available:
1.Manual
2.Semiautomatic
3.Automatic.
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B.DEVELOPING CHAMBER
• The "classical" flat-bottomed chamber is available
in many sizes from various manufacturers.
• It has two types:-
1.Twin Trough Chambers (TTC)
2. Automatic Developing Chamber (ADC)

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1. Twin Trough Chambers (TTC)
Twin trough chambers (TTC) (CAMAG) are among the most
widely used chambers.
• They are available in sizes10 *10 cm, 20 *10 cm, and 20 *20
cm.
• Only 5 ml of solvent is required per trough for HPTLC plate in
a 10 * 10 cm chamber.
2. Automatic Developing Chamber (ADC)
Here there is isocratic development of HPTLC plates.
• Steps like preconditioning of layer, chamber saturation,
development distance and final drying can be preset and
automatically monitored independent of environmental
effects.

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C. DERIVATIZATION DEVICE &
IMMERSION
DEVICES WITH PLATE HEATER
• Post chromatographic derivatization step.
• Substances that do not respond to visible or UV light can
be rendered detectable.
• Following are types:-
1. Immersion device
2. Auto reagent sprayer
3. Plate heater.

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IMMERSION DEVICE
• The chromatogram must be immersed.
• Withdrawn at a controlled uniform speed.
• Uniform vertical speed, freely selectable between 30
mm/s and 50 mm/s.
• Immersion time selectable between 1 and 8 seconds and
indefinitely.
• The device can be set to accommodate 10 cm and 20 cm
plate height.
• Battery operated, independent of power supply.

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AUTO REAGENT SPRAYER
• It is a low cost alternative for HPTLC/TLC.
• It contains a rubber pump but may also be operated
from a compressed air or nitrogen supply.

Plate heater.
• The TLC Plate Heater is designed for heating a
TLC/HPTLC plate to a selected temperature after a
staining reagent has been applied.

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D. SCANNING DENSITOMETER
• Chromatograms are evaluated densitometrically
• The photo sensor of the densitometer measures diffusely
reflected light.
• The difference between the optical signal from the
sample-free background and that from a sample zone
(fraction) is correlated with the amounts of the
respective fractions of calibration standards
chromategraphed on the same plate.
• Densitometry measurements of planar chromatograms
can be made by absorbance or fluorescence.

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APPLICATONS OF HPTLC
• Herbal fingerprinting
• Herbal Analysis – Quantification
• Pharmaceutical Science
• Determination of purity of sample and Identification of
compounds
• Identification of adulterants
• Forensic science
• Determination of mercury in water
• Analysis of environmental pollution levels
• Determination of ß-blockers like Metaprolol,
Alprenolol, Atenolol
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OTHER ACCESSORIES
i. Plate coater
ii. Drying rack
iii. Plate cutter.

QUALIFICATIONS (CAMAG)
• For customers working in a cGMP regulated environment,
CAMAG offers Installation Qualification
(IQ) and Operation Qualification (OQ) as service.

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Installation Qualification (IQ)
This qualification is performed at the site and time of
installation. It documents that all key aspects of the
installation comply with the manufacturer’s specifications,
codes, safety and design parameters. In order to qualify for
an IQ Certificate, this procedure is to be performed by a
Product Specialist, approved by company.

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 Operation Qualification (OQ)

• This qualification is performed subsequent to installation

and is repeated at certain interval recommended by the
manufacturer or defined by the customer. It documents
that all modules of the equipment perform consistently
throughout the specified operating ranges.
• The initial OQ is performed by the person responsible for
the IQ at installation. In order to qualify for an OQ
Certificate, this procedure is to be performed by a
Product Specialist, approved by company.
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Operation Qualification (OQ)…
• Repetitive OQ’s can be performed by a system user well
aquatinted with the system, following guidelines issued
by company. On request of the customer, such OQ’s can
be performed by a Product Specialist or Service
Engineer approved by company, against a fee or within
a service contract.
Performance Qualification (PQ)
• PQ is performed to ascertain that the instrument
(system) is suitable to perform a specific analytical task
as part of the manufacturing process.
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PQ…
• PQ is an on-going task with the customers samples and
procedures including preventive maintenance and
regular tests, such as system suitability and quality
control analyses with creation of QC-charts. For
computer systems it also includes regular data backup,
virus checks and change control procedures.
• PQ can thus only be performed by the user himself who
also has to create the SOP’s based on the analytical task,
the company OQ procedure, the different instrument
manuals and the customer's QC requirements.

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 Design Qualification (DQ)
• This qualification verifies that the rigorous specifications and
design review methods defined in the Quality Management
System of the manufacturer have been followed.
• Quality Management System ascertains planned testing
procedures, error reporting and controlled updating of
documents. Compliance is documented, e.g. by the
"Declaration of System Validation" and "Declaration of
Conformity" supplied with specific products.
• The Design Qualification is sometimes used in a different
meaning. One common misunderstanding is to use DQ for
"suitability of the laboratory equipment". To make sure the
laboratory is equipped with the necessary supporting
equipment etc.,
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QUALIFICATION OF LC-MS

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LIQUID CHROMATOGRAPHY
MASS SPECTROMETRY
•Liquid chromatography-mass spectrometry
(LC/MS) is a technique that uses liquid
chromatography (HPLC) with the mass spectrometry.
•It is an analytical chemistry technique that combines
the physical separation capabilities of liquid
chromatography with the mass analysis capabilities
of mass spectrometry.

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 PRINCIPLE
•The LC-MS technology involves use of an HPLC, wherein
individual components in a mixture are first separated followed by
ionization and separation of the ions on the basis of their
mass/charge ratio. The separated ions are then directed to a photo
multiplier tube detector, which identifies and quantifies each ion.
The ion source is an important component in any MS analysis, as
this basically aids in efficient generation of ions for analysis. To
ionize intact molecules, the ion source could be APCI
(Atmospheric Pressure Chemical Ionization), ESI (Electronspray
Ionization), etc. to name a few popular ones. The choice of ion
source also depends on the chemical nature of the analyte of
interest i.e. polar or nonpolar.
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Instrumentation of LCMS

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Components of Mass Spectrometer :
1. Ion source, which can convert gas phase sample
molecules into ions.
Following are the most common ionization methods :
i. Electrospray Ionization
ii. Atmospheric Pressure Chemical Ionization
iii. Atmospheric Pressure Photo-ionisation

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 Components of Mass Spectrometer :
2. Analyzer, where ions are separated according to their mass-to-charge ratio
by applying electromagnetic fields.
•Its task is to separate ions in terms of their mass-to charge
•Ratio and to direct the beam of focused ions to the detector.
•The key performance parameters of an analyzer include;
(a) separation efficiency
(b) m/z measurement precision
(c) range of the m/z values measured
•There are following kinds of mass analyzers that can be used in LC/MS :
(a)Quadrupole Analyzer,
(b)Time-of-Flight Analyzer,
(c) Ion Trap Analyzer
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 Components of Mass Spectrometer :
3. Detectors :
The detector is used to count the ions emergent from the
mass analyzer, and may also amplify the signal
generated from each ion.
Following are three different kinds of detectors are used
in Mass Spectrometry;
(a) Electron Multipliers
(b) Dynolyte Photomultiplier

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 Tandem Mass Spectrometry
•Tandem mass spectrometry (MS/MS) is a system of two
combined analyzers of the same type or different types,
characterized by high separation efficiency.
• The ions produced by the source are separated in the first
analyzer (MS1). Ions with the selected m/z value reach the
collision cell where, depending on the analysis conditions,
they undergo dissociation or remain unchanged.
• In comparison with analysis using a single analyzer,
tandem analysis shows a considerable improvement in
selectivity and considerably increased sensitivity.
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 OQ—
—Operational Qualification
• Temperature accuracy and stability of column heater/cooler
• Holmium oxide wavelength scan (if applicable)
• Detector lamp intensity and wavelength accuracy
• Detector noise and drift
• Pump flow rate accuracy and repeatability
• High and low pressure shutdown accuracy
• Injector precision
• Detector linearity and sample to-sample carryover
• Injection volume linearity
• Gradient composition accuracy
• Linear gradient tested using IPA and 0.5% Acetone/IPA
• Five step gradient tested using IPA and 0.5% Acetone/IPA
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IQ—Installation Qualification
• Inventory of instruction manuals,
• components and serial numbers
• Installation verification

IQ—Installation Qualification
• Inventory of instruction manuals,
• components and serial numbers
• Installation verification

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 Tests for HPLC Systems (Non-MSD)
Test Name Setpoints and Limits
Parameters

Vacuum N/A High vacuum min.: 8E-6 torr

Verification (any source)
High vacuum max.: 4E-5 torr
(any source),

Scan ůůƵƐĞĚŵĂƐƐĞƐ͗͸ϯ͘ϬƉƉŵ
Verification (DSES, ES+AJST;
N/A for others)

Response Evaluated mass: 156 Coefficient of determination

Linearity m/z Injection volume ;ƌϮͿ͗шϬ͘ϵϴϬϬϬ
on column: 20 ul* (DSES, ES+AJST; N/A for
others) 297
Tests for HPLC Systems (Non-MSD)
Test Name Setpoints and Parameters Limits

Injection Evaluated mass: 156 m/z ƌĞĂZ^͗чϮϬ͘ϬϬй;ĂŶLJ

Precision Injection volume on source)
column: 20 ul* Height RSD: Not applicable
(any
source)

Injection Carry Evaluated mass: 156 m/z ƌĞĂΘŚĞŝŐŚƚĐĂƌƌLJŽǀĞƌч

Over Injection volume on 1.00 %
column: 20 ul* (DSES, ES+AJST;
N/A for others)

Signal to Noise Evaluated mass: 156 m/z ^ŝŐŶĂůƚŽŶŽŝƐĞ͗шϭϬ;^^͕

Injection volume on ES+AJST; N/A for others)
column: 20 ul*

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Tests for Mass Spectrometer Detectors of
LCMS:
MSD 1. Vacuum Verification
Rationale: A stable, high vacuum is required for high-
sensitivity mass spectrometry.
Procedure: Multiple readings of the vacuum system are
taken and an automated comparison of these values to the
known acceptable values is made. Passing this test is a pre-
requisite for the following tests.
MSD 2. Scan Verification
Rationale: Calibration of mass range is critical in qualitative
mass spectrometry.

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MSD 2. Scan Verification…
Procedure: [Agilent LCMS] The built-in Agilent autotune is
performed to determine the proper calibration of the MSD and
ensure that masses are correctly reported across the entire mass
range of the instrument. [Non- Agilent LCMS] A manual tune is
made where applicable.
MSD 3. Response Linearity
Rationale: Knowledge of the response curve is critical for
quantitative analysis. Procedure: A sulfa drug mix standard of four
sulfonamide drugs is injected into the system at five concentrations
representing a wide range for LCMS.
The ions monitored are appropriate to the system type. The
calculated RSQ best-fit regression line and plot of the response
curve provides the statistics required to evaluate the instrument’s
overall response curve. This allows users to set appropriate
calibration ranges and limits in their quantitative application
methods. 300
MSD 4. Injection Precision
Rationale: System precision is critical for accuracy of quantitation.
Autosampler performance and MS ionization contribute to LCMS
system precision. Autosampler precision is challenged in the
standard LC module tests using a UV detector. A repeat precision
test in MS mode further challenges the precision of source
ionization and MS detection. Procedure: A blank injection followed
by six repeat injections of the sulfa drug mix followed by a final
blank injection are made. The %RSD of the six injections is
calculated to provide precision statistics.
MSD 5. Carry Over
Rationale: Low carry over from a previous injection is critical for
accuracy of quantitative and reliability of qualitative analysis.
Autosampler performance and MSD condition contribute to LCMS
carry over. Autosampler carry over is challenged in the standard LC
module tests using a UV detector.
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MSD 5. Carry Over
A repeat carry over test in MS mode further challenges the full
LCMS system carry over performance.
Procedure: A blank injection followed by single injection of the
highest concentration standard followed by a blank injection. The last
blank injection is evaluated for carry over and the result expressed as
a percentage of the value for the standard injection.

MSD 6. Signal to Noise

Rationale: Sensitivity of MS detection is an important performance
feature in quantitative and qualitative analysis. A signal-to-noise
value of representative compounds and appropriate ions at known
concentration provides sensitivity statistics.
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Reference
• https://www.slideshare.net/ankush96/qualification-of-
hptlc
• https://www.slideshare.net/ankush96/qualification-of-
lcms
• https://sciex.com/Documents/service/LCMS_Performance
_Qualification_svc.pdf

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