Under the guidance of

Dr. N. Usha Rani Ph. D

Department of Pharmaceutical Analysis

Maharajah’s college of Pharmacy

CONTENTS:

• Introduction.

• Principle.

• Detector response.

• Linearity range.

• Applications

1. Impurity control.

2. Assay determination.

3. Other Applications in pharmaceutical and related

areas.

• Conclusion.

• References.

INTRODUCTION:

M.S. Tswett (1903)– Liquid Chromatography (LC).

HPLC – wide spread technique.

UV/Vis Detector – widely used for its

. Sensitivity,

. Linearity,

. 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.

(1978)

DETECTORS LIMITATIONS

• UV/Vis Detector.

• Fluor metric Detector.

• RI Detector.

• Post column derivatization

UV detection.

•Amperometric Detector.

• ELSD.

• 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.

DETECTOR LIMITATIONS:

Dixon and Peterson - Corona – Charged Aerosol Detector

(CAD).

Allen et al - Nano Quantity Analyte Detector

(NQAD)

- Partly based on Condensation

Nucleation Light Scattering Detector

(CNLSD).

CAD is superior to ELSD and some extent to NQSD.

PRINCIPLE:

EAA – for sizing aerosols.

EAA operates by charging particles, pass near positive corona

discharge.

Small particles – large mobility, removed through negative charge.

Remaining charged particles, detected with Electrometer.

Advantages:

Aerosol charging is more sensitive to small particles.

Charge imparted to particles depends on particle size, but not on

particle composition.

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.

DETECTOR RESPONSE:

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

quantification.

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.

LINEARITY RANGE:

CAD response is not directly linear over a broad concentration

range.

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.

APPLICATIONS:

1. Impurity control.

2. Assay determination.

3. Applications in pharmaceutical and related areas.

IMPURITY CONTROL

1.Aminoglycosides.

2.Paclitaxel.

3.Maillard reaction.

4.Aminoacids.

5.Neuromuscular blocking drugs.

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

2gm.

Impurities exceeding this threshold must be quantified and

reported.

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.

1. AMINOGLYCOSIDES:

Identification and control of impurities by UV - Spectrophotometry,

mass spectrometry and pulsed amperometric detection (PAD).

METHOD DISADVANTAGE

• UV – Spectrophotometry

• Mass spectrometry

• Pulsed amperometric

detection (PAD).

• 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

volatile.

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.

2. PACLITAXEL:

Tetracyclic substance with heptacarbon skeleton and 11

stereocenters.

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

determined.

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 –

“Maillard reaction”.

Degradation products from MR, reported in pharmaceutical

products.

Determine MR impurities using HPLC – CAD, by Hydro – RP

column and mobile phase was capable of separating 4 MR

impurities.

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

0.03%.

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.

ASSAY DETERMINATION

1.Statin drugs.

2.Antidiabetic drugs.

3.Vitamins.

4.Gabapentin

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

210nm.

PRECISION – both UV and CAD more precise than ELSD.

CAD 5 fold more sensitive than ELSD, 3 times more sensitive

than UV.

LoQ – 16ng on column.

3. VITAMINS:

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.

4. GABAPENTIN:

No suitable chromophor.

Jia et al. developed method without derivitization prior to

analysis.

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

COUNTERIONS:

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

impurities.

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.

II. HYDROXYPROPYLMETHYLCELLULOSE:Widely used as tablet binder, film-coating, extended release tablet

matrix, capsule shell and thickening agent in ophthalmic

preparations.

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

liquid chromatography.

First dimension – RP-HPLC was applied.

- analyze the degree of

substitution.

Second dimension – SEC was applied.

- analyze molecular weight

distribution.

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

less pronounced.

CAD , a very useful detector for cleaning validation, especially in

low UV-response.

CONCLUSION:

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

analysis.

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