Simultaneous detection of nitrosamines and other sartan-related impurities in active pharmaceutical ingredients by supercritical fluid chromatography

Topic: Simultaneous detection of nitrosamines and other sartan-related
impurities in active pharmaceutical ingredients by supercritical fluid
chromatography.
Manipal college of pharmaceutical science
1 07-06-2023
Prepared by:
Mr. Sonawane Tejas
Registration No: 220609018
M Pharm, Semester III
Guided By :
Dr. Gundawar Ravi,
Assistant professor ,
Department of pharmaceutical quality
Assurance
MJC 302P
Journal Club In Pharmaceutical Quality Assurance

Article Details
Article title: Simultaneous detection of nitrosamines and other sartan-related impurities in
active pharmaceutical ingredients by supercritical fluid chromatography
Authors :Sebastian Schmidtsdorff , Alexander H. Schmidt
Journal: Journal of Pharmaceutical and Biomedical Analysis
Published on - 29 May 2019
Scopus indexed article (Q1)
 H-Index -141
Impact factor:3.571
DOI No.: https://doi.org/10.1016/j.jpba.2019.04.049 0731-7085
07-06-2023 Manipal college of pharmaceutical science 2

Contents :
• Introduction
• Literature Review
• Objective Of Study
• Material and Method
• Result
• Weakness
• Conclusion
• Future prospective
• Reference

Introduction
• Using the latest and innovative Quality-by-Design
(QbD) approach, it has now been possible to develop
an analytical method that enables to investigate
sartans, such as valsartan and losartan.
• Also a large class of different nitrosamines in the ppb
range and sartan-related impurities can thus be
determined simultaneously in a single analysis using
supercritical fluid chromatography (SFC).
• By using SFC, a broad spectrum of nonpolar and very
polar impurities can be separated and analyzed in
under 20 min.

Literature Review
Alkhateeb FL. Analytical Quality-by-Design Based Method Development for the Analysis of Formoterol,
Budesonide, and Related Compounds Using UHPLC-MS.
Liao X, Bei E, Li S, Ouyang Y, Wang J, Chen C, et al. Applying the polarity rapid assessment method to
characterize nitrosamine precursors and to understand their removal by drinking water treatment processes.
Water Res. 2015 Dec;87:292–8.
Alkhateeb FL. Automated Peak Tracking Using Mass Detection and Fusion QbD Software.
Automated Peak Tracking Using Mass Detection and Fusion QbD Software.pdf.
Angrish P, Technologies A. Nitrosamine Impurities Application Guide.

• Valsartan and
losartan with
their impurities
6/7/2023 Manipal college of pharmaceutical science 6

Nitrosamine Impurity
 Nitrosamine impurities became a focus for authorities in
July 2018, when they announced a recall of angiotensin
II receptor blocker (ARB) medicines, known as
“sartans”, due to the presence of an impurity, (NDMA).
Valsartan and Losartan are the worst affected and
several lots of these products have been recalled .
 Nitrosamine impurities may increase the risk of cancer
if people are exposed to them above acceptable levels .
 There are part of a group of high potency mutagenic
carcinogens referred to as the “cohort of concern” in
ICH M7 .

Supercritical Fluid
chromatography
• Principle – Adsorption and
partition chromatography
• Fluid is forced to temperature and
pressure above its critical point
• Higher Efficiency
• Faster speed of Analysis
• Efficiently Separate thermolabile
compounds

Objective of study
• The aim of this work is therefore to develop a
new analytical method under Quality-by-Design
(QbD) principles, which is capable of replacing
conventional purity methods for the
determination of related substances in sartans.
• Detecting different classes of NAs in trace
amounts, so that the release of the drug substance
can be implemented using a single and fast
technique .
• Helping laboratories to identify the appropriate
tools needed for accurate and confident analysis.

Material and
Method
Quality-by-Design development strategy and risk
assessment
Chemicals and reagents
Instrumentation and software
Chromatographic conditions and screening procedure
Spectroscopic and spectrometric conditions

Quality-by-Design development strategy and risk assessment
critical quality attribute monitored or controlled to ensure the process produces the desired
quality” .
The core parameters for development and optimization:
CQA - peak resolution , peak height and signal-to-noise , Retention time .
CPP - modifier and additives to mobile phase , column temperature and gradient
flow rate .
The predefined objectives and targets were also called “Analytical Target Profile” (ATP) . The
ATP of the method to be developed should be that
(a) all impurities are separated so that they can be detected individually
(b) the method is sufficiently sensitive for all impurities
The U.S. Environmental Protection Agency (EPA) considers “consuming up to 96 ng
NDMA/day is reasonably safe for human ingestion.
Threshold of Toxicological Concern” (TTC),

Chemicals and reagents
• Certified reference standards of the analytes
valsartan, losartan potassium,
• 5000 g/ml reference stock solution of NDMA
• 200 g/ml reference stock solution of NDiPA in
methanol (MeOH)
• Carbon dioxide N45 (99.995%) and nitrogen
N50 (99.999%) .
• For screening experiments HPLC-grade
solvents and reagents were used . MeOH , EtOH
, IpOH , ACN .
• For Optimization 0.1% TFA , formic acid

Instrumentation and
software
Chromatographic analysis was performed
using an Acquity UPC2 SFC system
Fusion QbD software (Version 9.8.1.199 -)
was utilized for multivariate data analysis
and method screening, by full automatized
DoE planning and construction.
Chemical structures and logP values were
generated by hyperchem ChemDraw
software .

Chromatographic conditions and screening procedure
• First attempt all columns were screened with a generic gradient 20 min, At a flow rate of 1.5 ml/min
at 25 0C and 500 C column temperature.
• Valsartan and losartan were injected as a standard solution containing 0.5 mg/ml each and NDMA as
a 10 g/ml standard solution.
• Peak shape, retention behavior and peak resolution was analyzed and the four best columns were
chosen.
• Then the nitrosamine mixture was injected as a 10 g/ml standard solution
• Screening two HSS C18 SB columns were connected in series and the flow rate was adjusted to 1.2
ml/min at 1800 psi`

Cont….
Manipal
college
of
pharmaceutical
science
• Additives were added to MeOH: formic acid (FA), acetic acid
(AcOH),trifluoroacetic acid (TFA); also 30% ammonia solution
(NH3) and triethylamine (TEA) for mobile phase screening and
10 mm ammonium formate .
• Four modifiers were screened: MeOH, ethanol (EtOH),
isopropanol (IpOH) and acetonitrile (ACN)
• Valsartan and losartan were spiked at the 0.5% level with all their
impurities and injected each as 10 mg/ml solutions in MeOH.
• Then the nitrosamine mixture was injected as a 10 g/ml API
sample directly dissolved in MeOH .
07-06-2023 16

Spectroscopic and spectrometric conditions
• Design ¨ Space of¨ modifier screening for NAs
generated by Fusion QbD software .
• Chromatograms were recorded from 190 to 400
nm at a scan rate of 10 Hz with
multiwavelength photodiode array detector
ande valuated at 230 nm .
• MS parameters were tuned by direct infusion of
a 1 g/ml standard solution of all NAs in MeOH
(containing also 0.1% formic acid).

Result
Screening
Optimization
Method validation

Screening
The method screening showed that only MeOH as eluent with an addition of TFA was able to
provide sufficient separation and of the NAs and sartans.
Eight screened SFC columns showed sufficient separation and retention behavior for losartan,
valsartan and NDMA.
These columns were selected and tested in order to evaluate, which column has the highest
retention and separation power for the NAs
To increase the separation performance for the subsequent modifier screening, two columns
were connected in series. Modifiers were systemically screened with the DoE support of
Fusion QbD software.
The visualized design space is displayed by a white area, were the predefined CQAs are used
to discriminate values of each CPP to maximize outcome

Optimization
• The amount of the modifier in the start, the column temperature (
60 °C) and the concentration of TFA in the modifier were further
optimized to generate a robust and highly efficient working point for
the NAs .
• Optimum separation and sensitivity was only achieved at 55 °C
column temperature
• Slightly smaller concentrations of TFA probably would have
improved ionization, but higher amounts were necessary to separate
the sartans.
• After the NAs had been separated with the highest possible sensitivity
and selectivity, the gradient for the elution was optimized for the
separation of all sartans .

Optimization
Manipal college of pharmaceutical science 6/7/2023 21
 Acceptable performance region
 T- target point
 Response –colour
 Unshaded region indicates Variable setting
combinations that now meet method performance
AND method robustness requirements.

• Optimization of make-up flow rate and
percentage of formic acid in MeOH – the
best results were obtained at 0.12 ml/min
and 0.35% formic acid
6/7/2023
23
(a) Nitrosamine Mixture (b)Valsartan (c)
Losartan each spiked with specified and
unspecified sartan impurity at 0.5 %level and
sample conc. 10 mg /ml
Detection found that :

Method Validation
 After the successful method development the procedure has been validated
according to the ICH Q2(R1) guideline .
 The detection limits were determined by deriving the signal-to noise ratios from the
measured linearity of a six-fold injection at ten levels for each substance.
 The values found in the study are therefore below the TTC and have to be
considered uncritical. In contrast, in a random sample investigation
 The limits of the impurities of nitrosamine established by FDA and EMA they were
slightly below the to acceptance criteria .

Weakness
 Isomers of Nitrosamine impurity was not
detected
 The retention time , Low sensitivity
of the nitrosamine impurities .
 Ionization affects the separation
 GC is the headspace method but they are
not able to detect volatile nitrosamine in
the sartans .

Conclusion
The study showed that it is possible to incorporate state-of-the-art analytical techniques for the purity
analysis of sartans.
The developed method is the first approach utilizing SFC to analyze NAs and shows the high
potential of this technique.
The sensitivity of the developed method is comparable to the published LC- and GC–MS/MS
methods by FDA and EMA .
By using a systematic QbD development approach, contaminants and potentially carcinogenic NAs
can be separated and detected simultaneously.
This results in a sensitive method, which can detect NAs in the picogram to femtogram range on
column.

Future prospective
 In a next step, to investigate other
non-sartans for NAs and modify
another detector that far exceeds the
selectivity of the mass spectrometer,
to detect NAs in even smaller
amounts and prove the advantages of
SFC technology for pharmaceutical
quality control.
 Additionally, further NAs will be
integrated to generate a complex and
comprehensive group monograph for
nitrosamines.

Reference
Schmidtsdorff S, Schmidt AH. Simultaneous detection of nitrosamines and other sartan-related
impurities in active pharmaceutical ingredients by supercritical fluid chromatography. Journal of
pharmaceutical and biomedical analysis. 2019 Sep 10;174:151-60.
07-06-2023
28

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