Application Note
Pharmaceuticals

Highly Sensitive LC/MS/MS Method
for the Simultaneous Quantification of
Mutagenic Azido Impurity Analogues
in Five Different Sartan APIs andFormulations
Detection of regulated genotoxic impurities from thedrug manufacturing process
Authors
Kartheek Srinivas Chidella,
Saikat Banerjee, andKannan Balakrishnan
Agilent Technologies, Inc.

Abstract
In 2018, the first incident of contamination of a potential genotoxic nitrosamine
impurity N-nitrosodimethylamine (NDMA) was reported in the angiotensin II
receptor blocker (ARB) drug Valsartan. This later expanded to the addition of more
nitrosamine impurities in various ARB category drug substances and products,
which lead to multiple product recalls by the US-FDA and EMA agencies. Anotherknown mutagenic azido impurity 5-(4'-(azidomethyl)-[1,1'-biphenyl]-2yl)-1H-tetrazole(AZBT) triggered multiple recalls by various regulatory agencies such as EMEA, UK
MHRA, Health Canada, and TGA for various sartan tetrazole ring therapeutics, whichcontain drugs such as Irbesartan, Losartan, and Valsartan. The European Directorate
for the Quality of Medicines and Health Care (EDQM) also alerted its CEP holders to
perform the required confirmatory tests. Azido impurities can form as a byproductduring the synthesis of sartan active pharmaceutical ingredients (APIs) with atetrazole ring.
This application note describes a highly sensitive and selective method using
an Agilent 6470 triple quadrupole LC/MS for the simultaneous determination of
two azido impurities 5-(4'-(azidomethyl)-[1,1'-biphenyl]-2yl)-1H-tetrazole (AZBT)and 4'-(azidomethyl)-[1,1'-biphenyl]-2-carbonitrile (AZBC) in five different sartan
APIs and formulations, including Losartan, Valsartan, Irbesartan, Candesartan,
and Olmesartan.

Introduction
Azido impurities are known mutagenic
impurities, which need to be controlled
as per the ICH M7 guidelines regarding
safe human consumption. Formationof azido impurities is possible
during the synthesis of sartan active
pharmaceutical ingredients (APIs)
containing a tetrazole ring. The
formation of the tetrazole ring in sartans
is achieved by the reaction between
appropriate nitrile and azido groups,
which leads to the formation of azide
impurity byproducts at trace levels.
Several recalls of sartan therapeutics,
including Irbesartan, Valsartan,and Losartan were triggered by UK
MHRA and Health Canada due to thepresence of AZBT. EDQM also alerted
its CEP holders to perform the required
confirmatory tests. Regulatory agencies
such as EDQM, Swissmedic, and the
Taiwan FDA have published LC/MS/MS
methods for the determination of AZBTin different sartan APIs and formulations.
LC/MS/MS is an inherently selective
and sensitive analytical technique thatis well-suited to detect and quantify
trace‑level potential genotoxic impurities,
and is widely used in the pharmaceuticalindustry. Multiple reaction monitoring(MRM) is the mode of acquisition used
for quantitative analysis using triple
quadrupole instruments. This mode
selectively filters the precursor ion
and product ions of the compound ofinterest, thereby increasing the sensitivity
and selectivity of the analysis. The
Agilent Jet Stream (AJS) ionization
source used in this application note
works by using thermal gradient focusing
technology, which helps to increase
the sensitivity of the instrument toreach lower detection limits. Whileregulatory limits for AZBT impurities
are defined as 4.0 ppm with a LOQ of2

0.4 ppm, this study concluded thateven lower LOQs of 0.2 ppm for AZBT
impurities could be obtained using theAgilent 6470 triple quadrupole LC/MS.
AZBT is the main impurity of regulatory
concern, to be quantified in sartans,while 4'-(azidomethyl)-[1,1'-biphenyl]2-carbonitrile (AZBC) quantification
is for information purposes only, as
mentioned in the regulatory agencies
published methods.

azido impurity analogues AZBT and
AZBC in five different sartan APIs
and formulations, including Losartan,
Valsartan, Irbesartan, Candesartan,
and Olmesartan which are captured in
Figure1. Of the two methods publishedin this application note, method 1 refers
to the simultaneous quantification of
AZBT and AZBC in five sartan API anddrug formulations, and method 2 refers
to the quantification of AZBT only, in fivesartan APIs. Both methods differed interms of sample preparation with respect
to the API sample concentration. Liquid
chromatography and mass spectrometry
conditions remained the same forboth methods.

The LC/MS/MS methods described inthis application note were developed
using the highly sensitive 6470 triple
quadrupole LC/MS instrument.
This method was developed for thesimultaneous determination of both

A

C

B
N
N

N

N
NH

N

–
+ N
N

N

D

E

F

G

–
+ N
N

Figure 1. Chemical structures of (a) azido biphenyl tetrazole impurity, (b) azido biphenyl Carbonitrile
impurity, (c) Losartan API, (d) Valsartan API, (e) Irbesartan API, (f) Candesartan API, (g) Olmesartan API.

Chemicals and reagents
Sartan APIs and azido impurity
standards AZBT and AZBC were locallyprocured from pharmaceutical company.
Drug formulations were purchased fromlocal pharmacies. Other LC/MS‑grade
solvents (methanol, water) werepurchased from Honeywell (Charlotte,NC, USA). Formic acid was purchasedfrom Fluka (now of Honeywell).

Experimental
Method 1
Sample preparation for Sartan drug
APIs (API final sample concentration
2.5 mg/mL)
Losartan, Valsartan, and Olmesartan
drug API:
1. Accurately weigh 250 mg of drug API
into a 15 mL centrifuge tube.
2. Add 10 mL of sample diluent.
3. Vortex the solution for 1 minute,followed by 10 minutes of sonication
so that the API is completely soluble.
4. Transfer 100 µL of the prepared
solution into an HPLC sample vial andadd 900 µL of diluent.
5. Vortex the mixture a final time
before loading onto the LC/MS/MS
for analysis.
Irbesartan and Candesartan drug API:
1. Accurately weigh 250 mg of drug API
into a 15 mL centrifuge tube.
2. Add 10 mL of sample diluent.
3. Vortex the solution for one minute,followed by 10 minutes of sonication.
4. Centrifuge for 15 minutes at
4,500 rpm.
5. Transfer 100 µL of the supernatantsolution into an HPLC sample vial andadd 900 µL of diluent.
6. Vortex the mixture a final time
before loading onto the LC/MS/MS
for analysis.

Sample preparation for Losartan
100 mg, Valsartan 160 mg, andIrbesartan 300 mg drug products(API final sample concentration
2.5 mg/mL)

6. Filter the supernatant solution using
a 0.22 µm PVDF membrane, andtransfer 100 µL into an HPLC sample
vial, followed by the addition of
900 µL of diluent.

1. Using the labeled concentration,
crush enough tablets to obtainat least three times the API
target weight.

7. Vortex the mix a final time before
loading onto the LC/MS/MS
for analysis.

2. Find the average weight per tablet
and accurately weigh the equivalentof 250 mg API.
3. Transfer to a 15 mL centrifuge tube
and add 10 mL of diluent.
4. Vortex for one minute, followed by
40 minutes of shaking using a shaker.
5. After extraction, centrifuge thesamples at 4,500 rpm for 15 minutes.
6. Filter the supernatant solution using
a 0.22 µm PVDF membrane, andtransfer 100 µL into an HPLC sample
vial, followed by the addition of
900 µL of diluent.
7. Vortex the mix a final time before
loading onto the LC/MS/MS
for analysis.

Sample preparation for Candesartan
32 mg and Olmesartan 40 mg
drug products (API final sample
concentration 1.25 mg/mL)
1. Using the labeled concentration,
crush enough tablets to obtainat least three times the API
target weight.
2. Find the average weight per tablet
and accurately weigh the equivalentof 125 mg API.
3. Transfer to a 15 mL centrifuge tube
and add 10 mL of diluent.
4. Vortex for one minute, followed by
40 minutes of shaking using a shaker.
5. After extraction, centrifuge thesamples at 4,500 rpm for 15 minutes.

Method 2
Sample preparation for Losartan,
Valsartan, Irbesartan, Candesartan,
and Olmesartan drug APIs (API final
sample concentration 0.3 mg/mL)
1. Accurately weigh 30 mg of drug
substance into a 15 mL centrifuge
tube and add 10 mL of diluent.
2. Vortex for one minute, followed by
10 minutes of sonication to ensurecomplete solubility.
3. Transfer 100 µL into an HPLC sample
vial and add 900 µL of diluent.
4. Vortex the mix a final time before
loading onto the LC/MS/MS
for analysis.

AZBT and AZBC preparation: standard
and calibration level
1. Accurately weigh 10 mg of each of
AZBT and AZBC.
2. Transfer each mixture into
10 mL volumetric flasks and add
100% methanol to obtain final
concentrations of stock 1 at
1,000 µg/mL.
3. Prepare a 10 µg/mL sample ofstock 2 for AZBT and AZBC byadding 100 µL of stock 1 to 9.9 mL of
100% methanol.
4. Further dilute stock 3 to 1 µg/mL byadding 1 mL of stock 2 to 9 mL of
100% methanol.
5. Prepare a series of calibration
standards from 1 µg/mL using
80% acetonitrile in water to obtain the
final concentrations of 50, 40, 20, 10,
5, 1, 0.25, and 0.1 ng/mL for AZBT,and 50, 40, 20, 10, 5, and 1 ng/mL
for AZBC.
3

Instrument parameters and setpoints

Data analysis

Table 1. UHPLC configuration and settings.

Data were acquired and analyzed using
Agilent MassHunter software version 10.
MS/MS transitions were obtained andoptimized using the Agilent MassHunter
Acquisition optimizer software to
determine optimal precursor and productions, fragmentor voltages, and collisionenergies upon injection of a neat solution
at a concentration level of 1,000 ng/mL,with 1 µL injection volume in flow
injection mode.

Parameter

Value

Instruments

Agilent 1290 Infinity II high speed pump (G7120A)
Agilent 1290 Infinity II multisampler (G7167B)
Agilent 1290 Infinity II multicolumn thermostat (G7116B)
Agilent 1290 Infinity II variable wavelength detector (G7114B)

Needle Wash

methanol/water (80/20)

Sample Diluent

acetonitrile/water (80/20)

Multisampler Temperature

15 °C

Injection Volume

5 µL

Analytical Column

Agilent InfinityLab Poroshell 120 PFP, 3.0 × 100 mm, 2.7 µm (p/n 695975-308)

Column Temperature

40 °C

Mobile Phase A

0.1% formic acid in water

Mobile Phase B

0.1% formic acid in acetonitrile/water (95/5)

Flow Rate

0.4 mL/min

Gradient

Time (min)
0
0.1
5.5
12
14
14.1
18

Stop Time18 minutes

Wavelengths

260 nm

%A
60
60
60
0
0
60
60

Table 2. Mass spectrometer configuration andsource settings.

Instrument

Agilent 6470 Triple
Quadrupole Mass
Spectrometer (G6470B)

%B
40
40
40
100
100
40
40

Flow (mL/min)
0.4
0.4
0.4
0.4
0.4
0.4
0.4

Table 3. Detailed MRM settings in MRM mode on the Agilent 6470 LC/TQ.
CompoundPrecursor Ion(m/z)

Product Ion(m/z)

AZBT (Quantifier)

278.2

235.1

50

AZBT (Qualifier)

278.2

207.1

50

AZBC (Quantifier)

207.0

179.1

AZBC (Qualifier)

207.0

151.0

Ion Source

Agilent Jet Stream (AJS)

MS/MS Mode

MRM

Ion Mode

Positive

Gas Temperature

300 °C

Gas Flow

6 L/min

Nebulizer Pressure

30 psiSheath Gas Heater

300 °C

Sheath Gas Flow

10 L/minNo.

Start Time(min)

Scan Type

Capillary Voltage,
Positive

4,000 V

1

0

MRM

MS

Nozzle Voltage

1,000 V

2

1.0

MRM

Waste

MS1/MS2 Resolution

0.7/0.7 (unit/unit)

3

3.2

MRM

MS

Dwell Time

50 ms

4

4.1

MRM

Waste

5

6.2

MRM

MS

6

8.2

MRM

Waste

7

16

MRM

MS

4

Table 4. Diverter Valve program used to divert all
sartan APIs to waste.
Diverter Valve

Dwell Time Fragmentor
(ms)
(V)

Collision
Energy (V)

CAV
(V)

Polarity

50

4

5

+

50

14

5

+

50

80

30

5

+

50

80

40

5

+

Results and discussion

Table 5. Representative S/N ratio data* for LOQ, LOD, and linearity data for both azido impurities AZBTand AZBC.

Method 1
Method development was
performed in positive mode using
an Agilent Jet Stream Source for the
optimization of mass spectrometry
parameters for AZBT and AZBC,
including precursor and product ions,capillary voltage, fragmentor voltages,
and collision energies to obtain thedesired sensitivity. Gas temperatures,gas flows, and dwell times were
optimized to establish the response
reproducibility. Chromatographic
separation was achieved between AZBT,AZBC, and all five sartans using thePoroshell PFP column.
Sample preparation was optimized interms of extraction time, and achieved
the desired recovery for AZBT in all fivesartan APIs and formulations Losartan,
Valsartan, Irbesartan, Candesartan,
and Olmesartan, and AZBC in Losartan,
Valsartan, Irbesartan, and Olmesartan
formulations only.
Critical parameters such as specificity,
reproducibility, linearity, recovery, LOQ,
and LOD are established.
LOQ and LOD limits and S/N values
are captured in Table 5. Reproducibility
data, including bracketing standards
at a limit level of 4.0 ppm and LOQ
are captured in Tables 6 and 7. The
calibration concentrations ranged
from 0.1 to 50 ng/mL, with specificdetails mentioned in Table 5. R2
values are greater than 0.99 for bothAZBT and AZBC impurities, displaying
linear responses throughout theconcentration range.
A summary of recovery experiments
for AZBT and AZBC at the limit level infive Sartan APIs and at the limit leveland LOQ level in sartan formulations arecaptured in Tables 8, 9, and 10.

LOD

LOQ

Linearity Range

No.

Compound

ng/mL

ppm

LOD
(S/N)

ng/mL

ppm

LOQ
(S/N)

R2

ng/mL

ppm

1

AZBT

0.125

0.05

29.1

0.5

0.2

132.8

0.9955

0.1 to 50

0.04 to 20

2

AZBC

0.625

0.25

9.2

2.5

1

20.3

0.9993

1.0 to 50

0.4 to 20* S/N was calculated using the RMS algorithm, noise width (0.6 min), with reference selected as sample using
Agilent MassHunter Quantitative software version 10.

Table 6. Representative data for reproducibilityof the method at 10.0 ng/mL (4 ppm in relation
to an API concentration of 2.5 mg/mL), including
bracketing standards.

Table 7. Representative data for reproducibility ofthe method at LOQ 0.5 ng/mL (0.2 ppm) of AZBTand 2.5 ng/mL (1.0 ppm) of AZBC in relation to an
API concentration of 2.5 mg/mL.

NumberAZBT

AZBC

Number

AZBT

AZBC

1

69,035

2,703

1

3,814

744

2

68,732

2,609

2

4,171

884

3

70,275

2,391

3

4,061

801

4

3,687

756
818

Initial Replicates

BracketingStandard

Initial Replicates

471,3322,526

569,552

2,408

5

3,974

6

69,222

2,632

6

4,111

721

Average

3,969.7

787.3

STD DEV

186.0

59.6

%RSD

4.7

7.6

7

69,094

2,692

Average69,606.0

2,565.9

STD DEV

906.4

127.8

%RSD

1.3

5.0

Table 8. Summary of the recovery experiment in sartan drug
APIs at 10 ng/mL (4.0 ppm in relation to an API concentration of
2.5 mg/mL).
API Sample Concentration 2.5 mg/mL
Number

API Name

1

Recovery %

ng/mL

ppm

AZBT

Losartan

10

4.0

95.3

AZBC
80

2

Valsartan

10

4.0

106.9

101.4

3

Irbesartan

10

4.0

106.4

90

4

Candesartan

10

4.0

91.5

89.9

5

Olmesartan

10

4.0

97.4

95.3

Table 9. Summary of recovery experiment in sartan drug products at
10 ng/mL (4.0 ppm in relation to an API concentration of 2.5 mg/mL).
Recovery %NumberFormulation Name

ng/mL

ppm

AZBT

1

Losartan 100 mg

10

4.0

100.5

AZBC
99.7

2

Valsartan 160 mg

10

4.0

100.2

103.7

3

Irbesartan 300 mg

10

4.0

96.3

102.7

4

Candesartan 32 mg

10

4.0

88.8

NA*

5

Olmesartan 40 mg

10

4.0

99.2

108

* NA: Satisfactory recovery was not observed in Candesartan 32 mg tablets
for AZBC.

5

Figures 2, 3, and 4 show the
representative extracted ion MRM
chromatograms from the 6470 LC/TQ,
showing elution and separation of
azido impurities in standard and spiked
sartan APIs and formulations. DAD
chromatograms were acquired tobracket the retention times of higher
concentrations of all sartan APIs, which
need to be diverted to waste to avoid
mass spectrometer contamination.
These high concentration of sartan APIs
are diverted to waste using the inbuilt
diverter valve of the Agilent 6470 LC/MS
by creating a diverter valve program as
mentioned in Table 4.

Table 10. Summary of recovery experiment in sartan drug products at LOQ (0.2 ppm of
AZBT and 1.0 ppm of AZBC in relation to an API concentration of 2.5 mg/mL).
LOQ
Number

API Name

1

Recovery %*LOQ

Recovery %

ng/mL

ppm

AZBT

ng/mL

ppm

AZBC

Losartan

0.5

0.2

107.7

2.5

1

102.4

2

Valsartan

0.5

0.2

97.4

2.5

1

106

3

Irbesartan

0.5

0.2

99

2.5

1

97.9

4

Candesartan

0.5

0.2

87

2.5

1

NA**

5

Olmesartan

0.5

0.2

98.8

2.5

1

113.5

* Recovery experiment performed in duplicates in five different sartan APIs at 4.0 ppm,
sartan drug products at 4.0 ppm, and LOQ levels using 2.5 mg/mL API test concentration.
Chromatographic separation can be further optimized if any interference is observed informulation samples due to matrix components with impurities retention times.
** NA: Satisfactory recovery not observed in Candesartan 32 mg tablets for AZBC.

×104
Counts

1.5

1

1 2

2 3

3 4

4 5

5 6

1.0

6 7

7

6 7

7

6 7

7

6 7

7

6 7

7

TIC chromatogram

0.5
0Counts

×104
1.0 1

3.578

1 2

2 3

3 4

4 5

5 6

1 2

2 3

3 4

4 5

5 6

1 2

2 3

3 4

4 5

1 2

2 3

3 4

4 5

AZBT impurity
quantifier ion

0.8
0.6
0.4
0.2
0

Counts

×103
1
6

3.578

AZBT impurity
qualifier ion

4
2
0
×102
4 1

7.241

5 6

AZBC impurity
quantifier ion

Counts32
1×102
Counts

3

1

7.228

5 6

AZBC impurity
qualifier ion

2
1
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Acquisition time (min)Figure 2. Representative standard chromatogram of AZBT and AZBC at 10 ng/mL (4.0 ppm in relation to an API concentration of 2.5 mg/mL).

6

16

17

18

Response
Response

×103
5

2 3

3 4

4 5

5 6

6 7

7

6 7

7

6 7

7

TIC chromatogram

1

3.583

1 2

2 3

3 4

4 5

5 6

AZBT impurity

0
×102

Response

2

×103
2.5

×103
2.5

1

1 2

2 3

3 4

4 5

7.241

5 6

AZBC impurity
2.552
Losartan DAD chromatogram0* 4.434
Valsartan DAD chromatogram

0
×103

1.771

2.5

Irbesartan DAD chromatogram

Response0×103

Response

Response

1 2

0

Response

Response×104 1
1

×103* 9.5712

Candesartan DAD chromatogram

0
1.780

2.5

Olmesartan DAD chromatogram

0
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Acquisition time (min)Figure 3. Representative chromatograms of the chromatographic separation between AZBT and AZBC impurity standards, with all five sartans: Losartan,
Valsartan, Irbesartan, Candesartan, and Olmesartan.

7

×104
Irbesartan 300 mg spike TIC chromatogram
0×103
Irbesartan 300 mg spike DAD chromatogram
0×103
AZBT impurity
0
×102
AZBC impurity

2

×103
Candesartan spike TIC chromatogram

Response0×103
Candesartan spike DAD chromatogram
0×103
AZBT impurity0
×104
Valsartan spike TIC chromatogram
0
×103
Valsartan spike DAD chromatogram
0×103
AZBT impurity
0
0

1

2

3

4

5

6

7

8
9
10
Acquisition time (min)

11

12

13

14

15

16

17

Figure 4A. Representative chromatograms of standards AZBT and AZBC at 10 ng/mL (4.0 ppm in relation to an API concentration of 2.5 mg/mL) spiked into
sartan formulations Irbesartan 300 mg, Candesartan 32 mg, and Valsartan 160 mg.

8

×102

1

1 2

2 3

3 4

4 5

* 7.224

5 6

2

6 7

7

6 7

7

6 7

7

6 7

7

6 7

7

6 7

7

6 7

7

AZBC impurity

×104
2 1

1 2

2 3

3 4

4 5

5 6

Lorsartan 100 mg spike TIC chromatogram
0
2.557

×103

Lorsartan 100 mg spike DAD chromatogram
0
×104

1

3.587

1 2

2 3

3 4

4 5

5 6

AZBT impurity

Response0×102 1

1 2

2 3

3 4

4 5

1 2

2 3

3 4

4 5

* 7.241

5 6

AZBT impurity

2.5

×103

1

5 6

Olmesartan 40 mg spike TIC chromatogram
0
×103

1.780
Olmesartan 40 mg spike DAD chromatogram

0×103 1

3.570

1 2

2 3

3 4

4 5

5 6

AZBT impurity
0
×102 1

1 2

2 3

3 4

4 5

5 6

* 7.219

2

1

2

3

4

5

6

7

AZBC impurity

8

9

10

11

12

13

14

15

16

17

Acquisition time (min)Figure 4B. Representative chromatograms of standards AZBT and AZBC at 10 ng/mL (4.0 ppm in relation to an API concentration of 2.5 mg/mL) spiked into
sartan formulations Losartan 100 mg and Olmesartan 40 mg.

9The calibration curve for both azido
impurities demonstrated an accuracy
rate within 15% of the expectedconcentration. Calibration levels wereas shown in Table 5, and reproducibility
across all levels exhibited CVs
less than 15%. Figure 5 shows thecalibration curves generated from the6470 LC/TQ system.

All sartan APIs are completely soluble
using the sample preparation method
mentioned earlier for method 2, andchromatographic separation performed
using the same conditions as inmethod 1.
All critical parameters (reproducibility,
recovery, linearity, and LOQ) areestablished for AZBT in all fivesartan APIs.
Table 11. Representative data for reproducibilityof the method at 1.25 ng/mL (4 ppm in relation
to an API concentration of 0.3 mg/mL)
including bracketing standards.
Number

AZBT

1

8,437

Bracketing Standard

10

AZBT
y = 8469.532809x – 42.535188
R2 = 0.99550922

Responses

×104
1.4
1.2
1.0

B
AZBC
y = 314.073117x + 9.481312
R2 = 0.99937755

0.8
0.6
0.4
0.2
0
-2 0 2 4

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
Concentration (ng/mL)

Figure 5. Representative calibration curves for AZBT (A) and AZBC (B) from the Agilent 6470 LC/TQ
for all azido impurities dispersed throughout the chromatogram. The calibration curves used a 1/x 2
weighting factor.

Table 12. Summary of the recovery experiment in sartan
drug substances at 1.25 ng/mL (4.0 ppm in relation to an
API concentration of 0.3 mg/mL).
API Sample Concentration 0.3 mg/mL

Recovery %Number

API Name

ng/mL

ppm

1

Losartan

1.25

4.0

AZBT
99.7

2

Valsartan

1.25

4.0

115.9

3

Irbesartan

1.25

4.0

96.6

4

Candesartan

1.25

4.0

95.2

5

Olmesartan

1.25

4.0

95.1

Table 13. Representative S/N ratio data for LOQ and LOD for AZBT.
LOD

LOQ

8,763

NumberCompound

ng/mL

ppm

LOD (S/N)

ng/mL

ppm

LOQ (S/N) *

3

8,937

1

AZBT

0.033

0.11

3.5

0.1

0.33

13.9

4

9,172

5

9,104

6

8,899

2

Initial Replicates

A

4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

-4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
Concentration (ng/mL)

Method 2
Method 1 was developed for
simultaneous quantification of bothAZBT and AZBC impurities at 2.5 mg/mL
as an API test concentration, whereas
method 2 was developed for thequantification of AZBT only, using
0.3 mg/mL API concentration for
all five sartans: Losartan, Valsartan,
Irbesartan, Candesartan, andOlmesartan, with a limit of 4.0 ppm andLOQ of 0.4 ppm.

×105

Responses

Accuracy and reproducibility

7

8,877

Average

8,884.1

STD DEV

241.1

%RSD

2.7

* S/N was calculated using the RMS algorithm, noise width (0.6 min) with reference
selected as sample using MassHunter Quantitative software, version 10.

×103
2 1

1 2

2 3

1 2

2 3

1 2

2 3

3 4

4 5

5 6

6 7

7

3 4

4 5

5 6

6 7

AZBT impurity
quantifier ion

7

4 5

5 6

6 7

AZBT impurity
qualifier ion

7

TIC chromatogram

0
×103
1 1

3.626

0
×102
5

1

* 3.631

3 4

0
2.583

Response

×103

Losartan DAD chromatogram

2
0
×103
2

4.542

Valsartan DAD chromatogram

0
1.765

×103
2.5

Irbesartan DAD chromatogram

0
9.535

×103
2

Candesartan DAD chromatogram

0
1.767

×103
2.5

Olmesartan DAD chromatogram

0
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Acquisition time (min)Figure 6. Representative chromatogram of the chromatographic separation between AZBT at 1.25 ng/mL, and the five sartans at 0.3 mg/mL sample
concentration (Losartan, Valsartan, Irbesartan, Candesartan, and Olmesartan).

11

Conclusion

References

Two different methods have beendeveloped in terms of differing API
sample concentrations, and could
establish all critical parameters of
method performance for azido impurities
using an Agilent 6470 LC/TQ. This
study analyzed azido impurities at very
low concentration levels, demanded
by regulatory requirements. This study
also detected the presence of AZBT
impurities below the limit level in theanalyzed Losartan formulations, using
the developed method conditions.
This application note is intended todemonstrate the reproducibility andsensitivity of the 6470 LC/TQ in thedetection of azido impurities in the fivedifferent sartan APIs and formulations
Losartan, Valsartan, Irbesartan,
Candesartan, and Olmesartan.

1. https://database.ich.org/sites/
default/files/M7_R1_Guideline.pdf
2. https://healthycanadians.gc.ca/
recall-alert-rappel-avis/hcsc/2021/75715a-eng.php
3. https://www.tga.gov.au/alert/azideimpurity-sartan-blood-pressuremedicines
4. https://www.dcatvci.org/pharmanews/7286-uk-regulators-recallbatches-of-sartan-drug-for-azidoimpurities
5. https://www.edqm.eu/en/news/
risk-presence-mutagenic-azidoimpurities-sartan-active-substancestetrazole-ring
6. https://www.swissmedic.ch/swissmedic/en/home/news/
mitteilungen/intensivierteueberwachung-sartan-arzneimittel.
htmlmL
7. https://www.edqm.eu/sites/default/
files/medias/fichiers/OMCL_
Network/31-pv-185-genotoxicsubstances-sartans.pdf
8. https://www.edqm.eu/sites/default/
files/general-methodparametersazbt-lcms.pdf
9. Method of Test for Azido
Compounds in Sartan Drug
Substances - Test of AZBT https://www.fda.gov.tw/ENG/siteList.
aspx?sid=10360

www.agilent.com/chem
DE41065069
This information is subject to change without notice.
© Agilent Technologies, Inc. 2022
Printed in the USA, January 25, 2022
5994-4536EN