Determination of pharmaceutical counterions. In assessment of excipients. In pharmaceutical cleaning validation
Under the guidance of
Dr. N. Usha Rani Ph. D
Department of Pharmaceutical Analysis
Maharajah’s college of Pharmacy
• Detector response.
• Linearity range.
1. Impurity control.
2. Assay determination.
3. Other Applications in pharmaceutical and related
M.S. Tswett (1903)– Liquid Chromatography (LC).
HPLC – wide spread technique.
UV/Vis Detector – widely used for its
. Compatibility with gradient elution.
Alternative – Refractive Index (RI) detector,
Amperometric Detector and
Electro Chemical (EC) or post column
derivatization UV detection.
Charles worth – Evaporative Light Scattering Detector.
• UV/Vis Detector.
• Fluor metric Detector.
• RI Detector.
• Post column derivatization
• Amperometric Detector.
• Lack of chromophor.
• Lack of fluorophor.
• Not sensitive and not suitable for
gradient elution .
• Difficult to validate.
•Technically difficult to apply.
• Non – linear detector response, Non
–reproducible spike peaks.
Dixon and Peterson - Corona – Charged Aerosol Detector
Allen et al - Nano Quantity Analyte Detector
- Partly based on Condensation
Nucleation Light Scattering Detector
CAD is superior to ELSD and some extent to NQSD.
EAA – for sizing aerosols.
EAA operates by charging particles, pass near positive corona
Small particles – large mobility, removed through negative charge.
Remaining charged particles, detected with Electrometer.
Aerosol charging is more sensitive to small particles.
Charge imparted to particles depends on particle size, but not on
CAD detector have an 10 – fold increased sensitivity than ELSD.
Detection by CAD, solute is less – volatile than mobile phase.
Choice of organic modifier is larger.
CAD response does not depend on functional groups or moieties.
Advantage – screening for impurity where no structural
information is available.
CAD detector response depends on amount of organic modifier
present in mobile phase.
Disadvantages – Not possible to use single external standard for
Post column addition of organic modifier to isocratic methods
with highly aqueous mobile phase, to reduce baseline noise and to
increase detector sensitivity.
LOQ’s for CAD range between 1 and 240 ng on column.
CAD response is not directly linear over a broad concentration
Only reported to be linear over a limited range of about 2
magnitude in different studies.
This is sufficient for assay determination or determination of
impurities, when external standard is used.
1. Impurity control.
2. Assay determination.
3. Applications in pharmaceutical and related areas.
Key objective in the quality control.
In guideline Q3A(R) and ICH has set threshold of 0.05% for
impurities in daily dose below 2gm and 0.03% daily dose above
Impurities exceeding this threshold must be quantified and
Methods used for control of impurities must exhibit LOQ’s which
corresponds at least to reporting threshold.
Method employed for control of impurities in “substances for
pharmaceutical use” need to be sufficiently sensitive.
Identification and control of impurities by UV - Spectrophotometry,
mass spectrometry and pulsed amperometric detection (PAD).
• UV – Spectrophotometry
• Mass spectrometry
• Pulsed amperometric
• Lack of a suitable chromophor, its requires high
Analyte concentrations and a low detection wavelength
(about 200 nm).
• Requires suitable specific reference materials for the
quantification of the impurities.
• Technically very difficult and Non - oxidizable
compounds are not detected.
Methods developed for separation of impurities could be coupled
to MS detection.
Mobile phase compatible with HPLC – CAD methods must be
By using different types of columns and mobile phases to analyze
amino glycosides in pharmaceutical preparations.
LOQs – 4ng on column for streptomycin,
- 100ng on column for netilmicin sulphate.
Tetracyclic substance with heptacarbon skeleton and 11
Used in anticancer therapy.
Isolated from bark of the pacific yew tree.
By using curosil – PFP column and mobile phase, relative
response factors (RRFs) of paclitaxel – related impurities were
It is fast, convenient and accurate method to determine UV RRFs
of known and unknown impurities.
3. MAILLARD REACTION:
Memantine (primary amine) - Alzheimer’s disease.
Primary amine reacts with reducing sugars in complex pathway –
Degradation products from MR, reported in pharmaceutical
Determine MR impurities using HPLC – CAD, by Hydro – RP
column and mobile phase was capable of separating 4 MR
Detection by CAD allowed to quantification of impurities without
a chromophor at levels 0.02 – 0.03%.
4. AMINO ACIDS:
Widely used biological compounds.
Lack of chromophor, HPLC – UV method is fail to analyze and
purity control of amino acids.
By using HPLC – CAD and ion pair HPLC method for the purity
control of aspartic acid.
LOQs for the potential impurities were found between 0.006 and
5. NEUROMUSCULAR BLOCKING DRUGS:
E.g.: Atracurium, Cisatracurium, and Mivacurium.
HPLC – CAD method used for the determination of isomers and
its degrades of Neuromuscular blocking drugs.
Thermo C18 column and mobile phase were used to determine
Linear detector response range from 5 – 50μg/ml.
Accuracy and Precision were found for all samples range from
96.5 to 101.6%.
Lowest LoQ’s were obtained for the impurity laudanoside with
16ng on column.
1. STATIN DRUGS:
Statin drugs – treatment of hypercholesterolemia.
Natural – Lovastatin,
Semi-synthetic – Simvastatin, Pravastatin.,
Synthetic – Rosuvastatin, Fluvastatin….
Novakova et al. develop a HPLC – CAD method for separation
and determine a LoQ of 1ng on column, 2.5times more sensitive
than UV 238nm.
2. ANTIDIABETIC DRUGS:
Lee et al. compared the performance of 3 different HPLC
detectors (UV, ELSD and CAD).
ACCURACY – 3 detectors delivered acceptable results.
RECOVERY – CAD better than ELSD and Superior to UV
PRECISION – both UV and CAD more precise than ELSD.
CAD 5 fold more sensitive than ELSD, 3 times more sensitive
LoQ – 16ng on column.
No simple analytical method for determination of Ascorbic acid
and Dehydroascorbic acid was available.
Novakova et al. developed a new HPLC – CAD method based on
hydrophilic interaction chromatography (HILIC).
Accuracy and Precision were acceptable.
LoQ – 10 µg/ml for Ascorbic acid,
- 1 µg/ml for Dehydroascorbic acid.
No suitable chromophor.
Jia et al. developed method without derivitization prior to
Performance were compared on ELSD and CAD detectors.
LoD – 5-30ng for ELSD,
- 7.5-10ng for CAD, compared to 300 - 425ng for UV.
CAD 25 times more sensitive than ELSD.
APPLICATIONS IN PHARMACEUTICAL
AND RELATED AREAS
1.Determination of pharmaceutical counterions.
2.In assessment of excipients.
3.In pharmaceutical cleaning validation
1. DETERMINATION OF PHARMACEUTICAL
To obtain physicochemical properties, drug substances are
produced in the form of salt.
Salts can be formed during synthesis and remain in the product as
Zhang et al. developed CAD method for determination of 25 most
commonly used pharmaceutical counterions.
LoD – 5ng for Sodium,
- 400ng for Lactate.
Huang et al. developed a HILIC mode method to determine
several counterions in the presence of an API.
Method was superior to ion-chromatography.
2. IN ASSESSMENT OF EEXCIPIENTS:
i. Polysorbate 80.
ii. Hydroxypropylmethylcellulose (HPMC).
I. POLYSORBATE 80:
Non-ionic surfactant, heterogeneous compound, consisting of
oleic acid esters of sorbitol and its anhydrides copolymerized with
20 mole ethylene oxide.
HPLC-UV determination of oleic acid after alkalimetric
hydrolysis at 195nm.
Feteke et al. developed fast HPLC-CAD method for determining
polysorbate 80 containing proteins.
First main peak was used for its quantification.
Lobback et al. compared the performance of ELSD and CAD,
CAD was shown to be 10 times more sensitive.
Widely used as tablet binder, film-coating, extended release tablet
matrix, capsule shell and thickening agent in ophthalmic
several types of HPMC are available differing in the degree of
substituition and molecular weight distribution.
Greiderer et al. developed a method based on two-dimensional
First dimension – RP-HPLC was applied.
- analyze the degree of
Second dimension – SEC was applied.
- analyze molecular weight
Method was also capable to estimate ‘cloud point temperature’.
3. IN PHARMACEUTICAL CLEANING VALIDATION:
Methods used for cleaning validation are similar to methods used
for impurity control.
several common cleaning solvents for manufacturing equipment
spiked with typical APIs and excipients.
APIs LoQ’s – 2-5ng for CAD,
- 3-6ng for UV detection.
Compared with UV, the interference with cleaning solvents was
CAD , a very useful detector for cleaning validation, especially in
Currently CAD appears to be most powerful and
versatile detector based on its applicability.
A prerequisite for the use of CAD is, similar to MS
detection, the use of volatile mobile phases.
Hence its ease of use, make the CAD a powerful
detector for research purpose, method development and routine
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