Download 6. BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED OF THE

6. BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED OF THE STUDY:
Liquid Chromatography – Mass Spectrometry (LC-MS) is a hyphenated technique in which a HPLC system is used
along with mass spectrometer detector. The HPLC separates chemicals by conventional chromatography on a
column usually by reverse phase chromatography, where the metabolite binds to the column by hydrophobic
interactions in the presence of a hydrophilic solvent (for instance water) and is eluted off by a more hydrophobic
solvent (methanol or acetonitrile). As the metabolites appear from the end of the column they enter the mass
detector, where the solvent is removed and the metabolites are ionised. The metabolites must be ionised because the
detector can only work with ions, not neutral molecules. And ions only fly through a very good vacuum, so removal
of the solvent is a vital first step. The mass detector then scans the molecules it sees by mass and produces a full
high-resolution spectrum, separating all ions that have different masses.
Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next
influenza pandemic. Oseltamivir is an ethyl ester prodrug requiring ester hydrolysis for conversion to the active
form, oseltamivir carboxylate, which has high bioavailability and penetrates sites of infection at concentrations that
are sufficient to inhibit viral replication. The proposed mechanism of action of oseltamivir is inhibition of influenza
virus neuraminidase with the possibility of alteration of virus particle aggregation and release. Tamiflu, available in
both capsule and powder for oral suspension form has been developed for the treatment of both A and B strains of
the disease.
Tamiflu (oseltamivir phosphate) is available as capsules containing 30 mg, 45 mg, or 75 mg oseltamivir for
oral use, in the form of oseltamivir phosphate, and as a powder for oral suspension, which when constituted with
water as directed contains 12 mg/mL oseltamivir base.
The production method is validated to ensure that the substance is the (3R,4R,5S)-enantiomer and that less
than 100 ppm
of the
impurity ethyl-(2R,3R,4R,5S)-2-azido-4-acetylamino-5-amino-3-(1-ethyl-propoxy)-
cyclohexane-1-carboxylate is present, when determined by a suitable method such as liquid chromatography
combined with mass spectrometry. Where necessary, the production method is also validated to demonstrate that
not more than 0.1% of tributyl-phosphine oxide is present in the final product, when examined by a suitable method
such as gas chromatography (GC).
Advantages of LC-MS:
 Selectivity: Co-eluting peaks can be isolated by mass selectivity and are not constrained by chromatographic
resolution.
 Peak assignment: A chemical fingerprint for the compound of interest is generated, ensuring correct peak
assignment in the presence of complex matrices.
 Molecular weight information: Confirmation and identification of known and unknown compounds.
 Structural information: Controlled fragmentation enables structural elucidation.
 Rapid method development: Provides easy identification of eluted analytes without retention time
validation.
 Sample matrix adaptability: Decreases sample preparation time.
 Quantization: Quantitative and qualitative data can be obtained easily with limited instrument optimization.
Ionisation methods:
A mass spectrometer works by using magnetic and electric fields to exert forces on charged particles
(ions) in a vacuum. Therefore, a compound must be charged or ionized to be analyzed by a mass spectrometer.
Furthermore, the ions must be introduced in the gas phase into the vacuum system of the mass spectrometer. This is
easily done for gaseous or heat-volatile samples. However, many (thermally labile) analytes decompose upon
heating. These kinds of samples require either desorption or desolvation methods if they are to be analyzed by mass
spectrometry. Although ionization and desorption/desolvation are usually separate processes, the term "ionization
method" is commonly used to refer to both ionization and desorption (or desolvation) methods. The choice of
ionization method depends on the nature of the sample and the type of information required from the analysis.
The different techniques involved in the ionisation methods include:
1. Gas phase ionisation.

Electron Ionisation (EI)

Chemical Ionisation (CI)

Desorption Chemical Ionisation (DCI)

Negative-ion Chemical Ionisation (NCI)
2. Field desorption and ionisation.

Field Desorption (FD)

Field Ionisation (FI)
3. Particle bombardment.

Fast Atom Bombardment (FAB)

Secondary Ion Mass Spectrometry (SIMS)
4. Atmospheric pressure ionisation (Spray methods)

Electrospray Ionisation (ESI)

Atmospheric Pressure Chemical Ionisation (APCI)
5. Laser desorption.

Matrix-Assisted Laser Desorption Ionisation (MALDI)
Detectors used in mass spectrometry:
The simplest detector is a Faraday cup, i.e., an electrode where the ions deposit their charge. The
electric current flowing away from that electrode results in a voltage when passing through a resistor of high
impedance. Faraday cups are still in use to measure abundance ratios with highest accuracy in isotope ratio mass
spectrometry (IR-MS). In the era of Mattauch-Herzog type instruments, the photographic plate has been the
standard detection system. With the advent of scanning mass spectrometers, secondary electron multipliers (SEM)
became predominant. These rely on the emission of secondary electrons from surfaces upon impact of energetic
ions. Progress has been made to employ cryogenic detectors, a rather special type of an ion counting detector for
high-mass ions in TOF-MS. The first commercial cryogenic detector TOF instrument has only recently been
released (Comet Macromizer, Flamatt, Switzerland). Ion counting detectors are not used in FT-ICR-MS where
image current detection yields superior results.
The various detectors used are:
1. Discrete Dynode Electron Multipliers
2. Channel Electron Multipliers
3. Micro channel Plates
4. Post-Acceleration and Conversion Dynode
5. Focal Plane Detectors
Applications in drug development:
The combination of high-performance liquid chromatography and mass spectrometry has had a
significant impact on drug development over the past decade. Continual improvements in LC-MS interface
technologies combined with powerful features for structure analysis, qualitative and quantitative, have resulted in a
widened scope of application. These improvements coincided with breakthroughs in combinatorial chemistry,
molecular biology, and an overall industry trend of accelerated development. New technologies have created a
situation where the rate of sample generation far exceeds the rate of sample analysis. As a result, new paradigms for
the analysis of drugs and related substances have been developed. The growth in LC-MS applications has been
extensive, with retention time and molecular weight emerging as essential analytical features from drug target to
product. LC-MS based methodologies that involve automation, predictive or surrogate models, and open access
systems have become a permanent fixture in the drug development landscape. An iterative cycle of ``what is it?''
and ``how much is there?'' continues to fuel the tremendous growth of LC-MS in the pharmaceutical industry.
During this time, LC-MS has become widely accepted as an integral part of the drug development process.
Integration of LC-MS in drug development:
The significance is of two folds:
 LC-MS has, indeed, become the method of choice for many pharmaceutical analyses. Because the
utilization of analysis technology in the pharmaceutical industry is highly dependent on perception, the
breakthroughs and barriers that LC-MS has overcome provided opportunity for acceptance. Currently, LCMS is widely perceived in the pharmaceutical industry to be a viable choice, as opposed to a necessary
alternative, for analysis.
 These events led to an increased understanding of LC-MS in such a way that practitioners and collaborators
have become more diverse. The result of this diversity is a mutually shared sense of purpose within the
industry, inspiring creativity and generating new perspectives on analysis.
Future trends and perspectives:
LC-MS has proved to be an extremely sensitive and specific technique for the analysis of
pharmaceuticals. It plays important roles in the studies of drug metabolism, discovery of new drug candidates and
the analysis, identification and characterization of impurities and degradants in drug substances and products.
Technical advances in MS continue, with improvements in sensitivity and resolution. The trend is towards the
further development of hybrid instruments such as Q-TOF. FT-ICR will become more prominent as developments
are made and instruments become less complex and more available. The likely importance of proteomics in
pharmaceutical development will have implications for MS, leading to the further requirements for high resolution
sequencing. Coupling high-throughput sample preparation techniques with multiplexed LC-MS/MS will lead to
even faster analysis and the potential of interfacing LC-NMR with MS to give an LC-NMR-MS system will allow
the unequivocal identification of drugs and metabolites.
6.2 REVIEW OF LITERATURE:
Stability is an important pre-analytical variable for quantitative LC-MS/MS analysis of drug
molecules and/or their metabolites in biological matrices. Instability of an analyte in any stage of the bioanalytical
process, including sample collection, processing, storage, extraction and LC-MS/MS analysis, can result in
under/over-estimation if an adequate preventive procedure is not in place. In the current review on practical
strategies in quantitative LC-MS/MS bio-analysis of unstable small molecules, the common causes of analyte
instability were examined. The instability of some analytes is readily predictable because of the presence of certain
chemically or biologically labile moieties in the molecules or because the compounds are in an inter-convertible
form, e.g. lactone vs. hydroxyl carboxylic acid. However, the instability of many other analytes is not readily
predictable. Necessary evaluation needs to be conducted to identify the possible instability issues. The current
review highlighted some general considerations and specific approaches for developing a robust LC-MS/MS
method. In particular, incurred samples should be used as part of routine short-term stability assessment of any
unstable analyte during the early stages of method development and validation. This can help unveil any ‘hidden’
instability issues that, if left unaddressed, could lead to the in validation of a ‘validated’ method.1
The use of LC–MS for bioanalysis of pharmaceuticals is entering its third decade and may be
considered to be a mature technology. In many respects this is true, considering the advances made in such areas as
instrument performance, electronics, software and automation of use. However, there remain instrumental and
noninstrumental areas that require significant attention to ensure data quality. Increasing regulatory focuses on
analytical method performance and unaddressed method issues require the bioanalyst to understand those areas that
most greatly impact data quality.2
Oseltamivir phosphate is an ester pro-drug of the anti-influenza neuraminidase inhibitor, Tamiflu, has
been developed for the treatment of both A and B strains of the disease7. An HPLC–MS/MS assay method has been
developed for both compounds in plasma and urine which fulfills all of the criteria for a good analytical method. It
is sensitive with limits of quantification of 1 and 10 ng/ml for the pro-drug and active neuraminidase inhibitor
respectively. Extensive stability studies have demonstrated the absence of significant problems associated with the
decomposition of either compound.3
The different analytical methods include:

Colorimetric methods

Spectrofluorimetric methods

Enzymatic assay

Mass spectrometry methods

HPLC methods

Capillary electrophoresis methods
HPLC with tandem mass spectrometric detection was the most attractive option for the simultaneous assay
of oseltamivir (OP) and oseltamivir carboxylate (OC) in biological samples.4 Wiltshire et al. developed an HPLC–
MS/MS assay for both compounds in plasma and urine. After solid-phase extraction (SPE) procedure, the drug and prodrug were separated by RP-HPLC using a Nova-Pak CN HP cartridge housed in a radial compression module. The
mobile phase was 50% methanol – 50% 80 mM aqueous formic acid, pH 3. The flow-rate was 0.5 ml/min and the
retention times for the two compounds were approximately 3.5 (OC) and 5 min (OP). The analytes were introduced into
the mass spectrometer via the appropriate interface and the protonated molecular ions fragmented by collision with
argon. The resultant daughter ions were then detected.
Later, a method for the analysis of OP and OC in human plasma, saliva and urine using off-line SPE and
liquid chromatography coupled to positive tandem mass spectroscopy. OP and OC were analyzed on a ZIC-HILIC
column using a mobile phase gradient containing acetonitrile–ammonium acetate buffer (pH 3.5; 10 mM) at a flow rate
of 500 μL/min. The ZIC-HILIC column has a sulfobetaine type zwitterionic stationary phase covalently attached to
silica and is especially suitable for polar and hydrophilic analytes. The mobile phase should contain a high concentration
of water miscible organic solvent (e.g. acetonitrile) to promote hydrophilic and weak electrostatic interactions between
the analytes and the hydrophilic phase. Neutral and lipophilic compounds in general show poor retention on the column.
A triple quadrupole mass spectrometer with a TurboV ionisation source interface operated in the positive ion mode was
used for the multiple reactions monitoring LC–MS/MS analysis. This highly sensitive robust LC–MS assay should
facilitate urgently needed pharmacokinetic studies on this important antiviral drug.5
The
determination
of OP
and
OC
in rat plasma, cerebrospinal
fluid (CSF)
and brain and
in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry. Three fold
deuterated OP and OC served as internal standards. Protein precipitation with per chloric acid was followed by online SPE and gradient separation on a reversed-phase column. After electrospray ionization, the compounds were
detected in positive ion selected reaction monitoring (SRM) mode. Run time was 3.6 min. The lower limits of
quantification
(LLOQ)
were
0.1 ng/mL
in rat plasma and CSF,
0.5 ng/g
in brain and
1 ng/mL
in human plasma and urine. Inter-day and intra-day precisions and inaccuracies in rat matrices were below 10.2%
and 13.9% (below 19.0% at LLOQ), respectively. Intra-assay precisions and inaccuracies in human matrices were
below 11.7% and 8.9%, respectively. The recoveries were close to 100%, and no significant matrix effect was
observed. The method was successfully applied to rat study samples.6
A simple and rapid UV-spectrophotometer is the estimation method for the evaluation. Oseltamivir
is used in the treatment and prophylaxis of both influenza A and influenza B has been developed and assessed. The
proposed methods were successfully applied for the estimation of Oseltamivir in commercial pharmaceutical
preparation with UV detection at 208.5 nm. A Shimadzu 1700 U.V visible spectrophotometer with 1cm matched
quartz cells, and methanol solvent were employed in the method. Developed methods obeyed the Beer’s law in the
concentration range of 4-24μg/ml. methods were validated statistically. The SD of that was found 0.01125 and RSD
0.0210. The results of the tablet analysis were validated with respect to accuracy (recovery); linearity, limit of
detection and limit of quantitation were found to be satisfactory.7
In present investigation two simple and sensitive extractive spectrophotometric methods have been
developed and validated for the determination of Oseltamivir Phosphate in bulk and capsule dosage form. The
developed methods involve formation of extractable ion pair complex of drug with Bromocresol green and
Bromocresol purple dyes in acidic medium. Chloroform is used as extracting solvent for Bromocresol green and
Bromocresol purple. Extractable complexes shows maximum absorption at 416 nm and 404.5 nm and the drug
shows linearity range for Bromocresol green and Bromocresol purple in concentration range of 8-28 μg/ml. The
coloured chormophores were found to be stable for 24 hours.8
According to scientific research Anti-Influenza Prodrug Oseltamivir Is Activated by
Carboxylesterase Human Carboxylesterase 1 and the Activation Is Inhibited by Antiplatelet Agent Clopidogrel,
liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or
plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the
level of HCE1. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as
much as 90% when the equal concentration was assayed. Given the fact that hydrolysis of oseltamivir is required
for its therapeutic activity, concurrent use of both drugs would inhibit the activation of oseltamivir, thus making this
antiviral agent therapeutically inactive. This is epidemiologically of significance because people who receive
oseltamivir and clopidogrel simultaneously may maintain susceptibility to influenza infection or a source of
spreading influenza virus if already infected.9
6.3 OBJECTIVE OF STUDY:
The main objective of the study is to develop and validate an analytical method for oseltamivir
phosphate and its active metabolite to measure the concentrations in the available biological matrices. Bioanalytical
method validation is a procedure employed to demonstrate that an analytical method used for quantification of
analytes in a biological matrix is reliable and reproducible to achieve its purpose: to quantify the analyte with a
degree of accuracy and precision appropriate to the task. The quantitative approach used in bioanalytical methods
involves the use of a standard curve method with internal standard where the analyte concentration can be assigned
by referring the response to other samples, called calibrators or calibration standards. In addition to the samples of
unknown concentration, the bioanalytical set includes the calibration standards, and samples containing no analyte,
called blanks, to assure that there are no interferences in the matrix.
The data generated by these methods must be very reproducible and reliable because they are used
in the evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic, and preclinical findings. A
validation is required for all the bioanalytical methods employing analytical techniques such as gas chromatography
(GC), high-pressure liquid chromatography (HPLC), hyphenated mass spectrometric techniques such as LC-MS,
LC-MS/MS, GC-MS, and GC-MS/MS, or immunological and microbiological procedures such as
radioimmunoassay (RIA) and enzyme-linked immunosorbant assay (ELISA) for the quantitative determination of
drugs and/or metabolites in biological matrices such as blood, serum, plasma, urine, feces, saliva, sputum,
cerebrospinal fluid (CSF), tissues, and skin samples.
Although there are various stages in the validation of a bioanalytical procedure, the process by which a
specific assay is developed, validated, and used in routine sample analysis can be divided into four main steps:
1. Reference standards preparation (stock solutions, working solutions, spiked calibrators and QCs).
2. Bioanalytical method development where the assay procedure is established.
3. Bioanalytical method validation and definition of the acceptance criteria for the analytical run and/or batch.
4. Application of validated method to routine sample analysis.
An impressive variety of LC-MS based solutions, incorporating quantitative and qualitative process
approaches, are now routinely applied to accelerate drug development. The results are significant and led to the
successful development of innovative therapies and numerous novel drugs. The use of LC-MS with other
technologies for sample preparation, analysis, and data management is now an inextricably linked element of drug
development. The LC-MS applications indicate that this unique technology is now fundamentally established as a
valuable tool for analysis in the pharmaceutical industry, whereas new technologies continue to advance, the need
for information-rich tools such as LC-MS will continue to increase.
VALIDATION OF METHOD:
Assays of all samples of an analyte in a biological matrix should be completed within the time period
for which stability data are available. For a difficult procedure with a labile analyte where high precision and
accuracy specifications may be difficult to achieve, duplicate or even triplicate analyses can be performed for a
better estimation of analyte.
Following parameters are to be evaluated to ensure that the method is validated.
1. Specificity
2. Linearity
3. Range
4. Accuracy
5. Precision
a) Repeatability
b) Intermediate Precision
c) Reproducibility
6. Detection limit
7. Quantitation limit
8. Robustness
Once the analytical method has been validated for routine use, its accuracy and precision should be
monitored regularly to ensure that the method continues to perform satisfactorily. To achieve this objective, a
number of QC samples prepared separately should be analyzed with processed test samples at intervals based on the
total number of samples.
7. MATERIALS AND METHOD:
7.1 SOURCE OF DATA:
Data will be from the journals, USFDA, ICH Guidelines, WHO Guidelines, Internet, Science Direct, Articles, as
well as from e-library of Krupanidhi College of Pharmacy.
7.2 METHOD OF COLLECTION OF DATA:
The data will be collected on method development and validation indicating from standard books and literature
survey. The method developed is to be validated to check the variation in method conditions for robustness.
Data is obtained in the following types:

Raw data

Processed data

Interpreted data
Raw data reveals information about the drug development decision during the method development process of the
analytical method, whereas processed data and interpreted data reveal the integrated information about the method
refinement and method/process validation respectively.
Documentation for method development and establishment should include:

An operational description of the analytical method

Evidence of purity and identity of drug standards, metabolite standards, and internal standards used
in validation experiments

A description of stability studies and supporting data

A description of experiments conducted to determine accuracy, precision, recovery, selectivity, limit
of quantification, calibration curve (equations and weighting functions used, if any), and relevant
data obtained from these studies

Documentation of intra- and inter-assay precision and accuracy

In NDA submissions, information about cross-validation study data, if applicable

Legible annotated chromatograms or mass spectrograms, if applicable
7.3 DOES THE STUDY REQUIRE ANY INVESIGATIONS OF INTERVENTIONS TO BE
CONDUCTED ON PATIENTS OR OTHER HUMAN OR ANIMALS? IF SO PLEASE DESCRIBE
BRIEFLY?
No.
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTE IN CASE OF AS
ABOVE?
Not applicable
8. LIST OF REFERENCES:
1. Wenkui Li, Jie Zhang, Francis L.S. Tse. Strategies in quantitative LC-MS/MS analysis of unstable small
molecules in biological matrices. Biomedical Chromatography 2011;25:258–77.
2. William D. van Dongen. Bioanalytical LC–MS of therapeutic
oligonucleotides. Bioanalysis
2011;3(5):541-64.
3. Hugh Wiltshire, Barbara Wiltshire, Andrea Citron, Tony Clarke, Carolyn Serpe, Donald Gray, William
Herron. Development of a high-performance liquid chromatographic–mass spectrometric assay for the
specific and sensitive quantification of oseltamivir, an anti-influenza drug, and its pro-drug, oseltamivir, in
human and animal plasma and urine. J Chrom B 2000;745:373–88.
4. M. Espinosa Boscha, C. Bosch Ojedab, A.J. Ruiz Sánchezc, F. Sánchez Rojas. Research Journal of
Pharmaceutical, Biological and Chemical Sciences analytical methodologies for the determination of
oseltamivir. Research journal of Pharmaceutical, Biological and Chemical Sciences 2010;1(3):368-76.
5. N Lindegardh, W Hanpithakpong, Y Wattanagoon, P Singhasivanon, NJ White, NPJ Day. J Chrom B: Anal
Techno Biomed Life Sci 2007;859:74-83.
6. Katja Heinig, Franz Bucheli. Sensitive determination of oseltamivir and oseltamivir carboxylate in plasma,
urine, cerebrospinal fluid and brain by liquid chromatography–tandem mass spectrometry. J Chrom B 2008
Dec 1;876(1):7129-36.
7. Rauta CS, Ghargea DS, Dhabalea PN, Gonjaria ID, Hosmanib AH, Abhijeet H. Hosmani. Development and
validation of oseltamivir Phosphate by uv-spectrophotometer. International journal of PharmTech Research
2010;2(1):363-6.
8. Ashish Ashok Thatte, Pramila T. Simple Extractive Colorimetric Determination of Oseltamivir Phosphate
by Ion-Pair Complexation Method in Bulk and Capsule Dosage Form. International Journal of Research in
Pharmaceutical and Biomedical Sciences 2011;2(2):543-7.
9. Deshi Shi, Jian Yang, Dongfang Yang, Edward L. LeCluyse, Chris Black, Li You, “et al”. Anti-Influenza
Prodrug Oseltamivir Is Activated by Carboxylesterase Human Carboxylesterase 1, and the Activation Is
Inhibited by Antiplatelet Agent Clopidogrel. The journal of pharmacology and experimental therapeutics
(JPET) 2006;319:1477–84.
10. http://www.jic.ac.uk/services/metabolomics/topics/lcms/index.htm
11. http://www.scientistlive.com/European-Science-News/Chromatography/LCMS_detection%3A_powerful_tool_for_organic_compound_analysis/13865/
12. Mike S. Lee, Edward H. Kerns. LC/MS Applications in Drug Development. New York: John Wiley &
Sons;2002. P. 187-279.
13. Chang-Kee LIM, Gwyn LORD. Current Developments in LC-MS for Pharmaceutical Analysis. Biol.
Pharm. Bull 2002;25(5):547-57.
14. http://www.who.int/medicines/publications/pharmacopoeia/OseltamivirMono.pdf