Application Note
Pharmaceuticals

Simultaneous Quantitation of Four
Impurities in Clozapine API Using theAgilent 6470 Triple Quadrupole LC/MS
Quantitation of impurities 4-chloro 2-nitrobenzene
amine, 2-chlorobenzoic acid, 2-(4-chloro 2-nitro
phenyl amino) benzoic acid and 2-(2-amino 4-chloro
phenyl amino benzoic acid) in API (Clozapine)
AuthorPrasanth Joseph,
Saikat Banerjee, andKannan Balakrishnan
Agilent Technologies, Inc.

Abstract
The presence of potential toxic chemicals and impurities in drugs are one of thebiggest challenges in the manufacturing of active pharmaceutical ingredients (APIs).
Therefore, it is important to identify these impurities during the manufacturing
process to avoid issues related to the quality, efficacy, and safety of drugs. Thisapplication note describes an LC/MS/MS-based MRM method for the simultaneousquantitation of the four impurities 4-chloro 2-nitrobenzene amine, 2-chlorobenzoic
acid, 2-(4-chloro 2-nitro phenyl amino) benzoic acid, and 2-(2-amino 4-chloro phenyl
amino) benzoic acid in API (Clozapine). This application note demonstrates LOD-LOQ
determination of impurities based on the calibration curve method. The detection
limit is calculated as 3.3 × σ/S, whereas the quantification limit is calculated by
10 × σ/S, where σ is the standard deviation of the response, and S is the slope ofthe curve.
Quantitation of these impurities in clozapine API is extremely challenging due to thehigh matrix effect caused by the API, resulting in inaccurate quantitation. Therefore,chromatographic separation of the impurity from the API is necessary. The analysis
is made further complex, as the sensitivities of these four impurities are not similar.

4-Chloro 2-nitrobenzene amine(Impurity 2)
Clozapine (API)

Figure 1. Agilent 1290 Infinity II LC coupled to an Agilent
6470 triple quadrupole LC/MS.

Introduction
The presence of potential toxic chemicals and impurities indrugs is one of the biggest challenges in the manufacturingof API. Therefore, it is important to identify these impurities
during the manufacturing process to avoid issues related
to the quality, efficacy, and safety of drugs. Screening andquantitation of these impurities in APIs is useful to identify
potential problems when evaluating new suppliers, changing
manufacturing sites, or during production scale-up. An
LC/MS/MS method was developed for the simultaneousquantification of the impurities 4-chloro 2-nitrobenzene
amine, 2-chlorobenzoic acid, 2-(4-chloro 2-nitro phenyl amino)
benzoic acid, and 2-(2-amino 4-chloro phenyl amino) benzoic
acid in API for Clozapine.
In this application note, a highly selective multiple reaction
monitoring (MRM)-based LC/MS/MS method was developed
using an Agilent 6470 triple quadrupole LC/MS (LC/TQ). The
sensitivity of the 6470 LC/TQ can easily detect compounds at
the required limits of detection. The special design of the ion
optics and stable electronics of the system provide consistent
results across multiple batches.

Experimental
Chemicals and reagentsThe four impurities: 4-chloro 2-nitrobenzene amine,
2-chlorobenzoic acid, 2-(4-chloro 2-nitro phenyl amino)
benzoic acid, and 2-(2-amino 4-chloro phenyl amino) benzoic
acid were provided by a potential customer. LC/MS-grade
solvents such as methanol, acetonitrile, and water werepurchased from Honeywell (Charlotte, NC, USA). Formic acid,
MS grade was purchased from Fluka (now of Honeywell).

22-Chlorobenzoic acid
(Impurity 1)

2-(4-Chloro 2-nitro phenyl amino) benzoic acid
(Impurity 3)

2-(2-amino 4-chloro phenyl amino) benzoic acid
(Impurity 4)
Figure 2. Structures of Clozapine and four impurities.

Instrument configuration– 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 diode array detector (G7117A)– Agilent 6470 triple quadrupole LC/MS (G6470B)
Table 1. Chromatography conditions.
Parameter

Value

Mobile Phase A

0.1% Acetic acid and 1 mM ammonium fluoride in water

Mobile Phase B

Methanol (100%)

Flow Rate

0.5 mL/min

Injection Volume

20 µL

Column Temperature

50 °C

Needle Wash

Methanol/water (70/30)

ColumnAgilent Poroshell HPH C18, 4.6 × 150 mm,
2.7 µm (p/n 693975-702) (T)Table 2. Gradient.
Time (min)

%A

%B

0

95

5

2

95

5

5

40

60

8

25

75

10

25

75

10.2

0

100

13

0

100

13.1

95

5

Post run

2 minutes

Concentration levels ranging from 0.15 to 15 ppb wereprepared as explained in Tables 3.1 and 3.2. Calibration
curves were plotted to establish the LOD and LOQ levels. Limitof detection and limit of quantitation were calculated from theslope and standard error of the predicted y-value for each x in
the regression of the calibration curve.
Instrument MRM parameters (Table 4) and source
parameters (Table 5) were optimized to maximize sensitivity,
while maintaining consistency in the method performance forlarge batches.
Table 5. MS source parameters.

Preparation of working standards for
LOD-LOQ calculation

Parameter

Value

Ionization SouceAJS ESI

Gas Temperature

300 °C

Gas Flow

10 L/min

Nebulizer

30 psi150 ppb

Sheath Gas

250 °C

Sheath Gas Flow

10 L/minCapillary Voltage

3,500 V

Nozzle Voltage

500 V

Table 3.1. Preparation of working standards, set 1.
WorkingStandard
1 ppm

VolumeTaken

Volumeof Diluent

0.75 mL

4.25 mL

Total
VolumeResultant
Concentration

5.0 mL

1 ppm

0.60 mL

4.4 mL

5.0 mL120 ppb

150 ppb

1.0 mL

1.0 mL

2.0 mL75 ppb120 ppb

1.875 mL

3.125 mL

5.0 mL45 ppb150 ppb

1.0 mL

4.0 mL

5.0 mL30 ppb30 ppb

1.0 mL

1.0 mL

2.0 mL

15 ppb15 ppb

1.0 mL

1.0 mL

2.0 mL

7.5 ppb

15 ppb

0.5 mL

4.5 mL

5.0 mL

1.5 ppb

Once the chromatographic separation between the API and
impurities was established, the time program was set todivert the API to waste with the help of an integrated diverter
valve (Table 6).

100 µL of each of the diluters were pipetted and filled up to
1 mL with diluent.

Table 6. Diverter valve program.

Table 3.2. Preparation of working standards, set 2.

Start Time (min)WorkingStandard

VolumeTaken

Volumeof Diluent

Total
VolumeResultant
Concentration

150 ppb

0.1 mL0.9 mL

1.0 mL

15 ppb120 ppb

0.1 mL0.9 mL

1.0 mL12 ppb75 ppb

0.1 mL0.9 mL

1.0 mL

7.5 ppb

45 ppb

0.1 mL0.9 mL

1.0 mL

4.5 ppb

30 ppb

0.1 mL0.9 mL

1.0 mL

3.0 ppb

15 ppb

0.1 mL0.9 mL

1.0 mL

1.5 ppb

7.5 ppb

0.1 mL0.9 mL

1.0 mL0.75 ppb

1.5 ppb

0.1 mL0.9 mL

1.0 mL0.15 ppb

Scan Type

Diverter Valve0

MRM

To waste

8

MRM

To MS

Table 4. MRM parameters.
ID

Precursor Ion(m/z)

Product Ion(m/z)

Dwell Time(ms)

Fragmentor(V)

Collision Energy(V)

Cell Accelerator
Voltage (V)Polarity

Clozapine

327

270.1

50

147

24

4

Positive

Impurity 1

155

111

50

74

4

4

Negative

Inpurity 2

171

141

50

70

16

4

Negative

Inpurity 3

291

216

50

92

16

4

Negative

Impurity 4

261

217

50

45

16

4

Negative

3

Sample preparation
API preparation
20 mg of API was weighed in a centrifuge tube. One milliliterof 60/40 methanol/water was added and vortexed for almost
two minutes. The mixture was kept in an ultrasonic bath forfive minutes, and the API was partially dissolved. However,
the impurities have good solubility in the solvent systemused (60/40 methanol/water). The partial solubility of the
API is helpful in reducing the matrix effect. The contents
were then filtered through a PVDF filter into a 2 mL HPLC vial
for injection.

Calibration curves generated from 0.15 ng/mL (LOQ) to
15 ng/mL for LOD-LOQ calculation

Figure 3.1. Calibration curve generated for Impurity 1 from 0.15 ng/mL (LOQ)
to 15 ng/mL for LOD-LOQ calculation.

Data acquisition and data analysis
All samples were acquired using Agilent MassHunter Data
Acquisition software, version 10.1. MRM transitions wereobtained and optimized using the Agilent MassHunter
Acquisition optimizer software. This tool automatically
optimized Fragmentor voltages for the Q1 precursor ions, andcollision energies for the Q3 product ions.
A standard solution of concentration, 500 ng/mL, wasintroduced to the MS by Flow Injection Analysis with aninjection volume of 5 µL. Through the automated workflow,
10 product ions from each impurity were selected for creation
to MRM transitions.

Figure 3.2. Calibration curve generated for Impurity 2 from 0.15 ng/mL (LOQ)
to 15 ng/mL for LOD-LOQ calculation.

Chromatograms were viewed through MassHunter qualitative
analysis software, version 10.0. Quantitation of each batch
was carried out using MassHunter quantitative analysis
software, version 10.1.
Validation parameters such as linearity, reproducibility,
recovery, specificity, and sensitivity, in terms of limit ofquantification (LOQ) and limit of detection (LOD), werecharacterized to ensure good method performance.
Accuracies for calibration points were within ±20%.
No manual integration was needed.

Figure 3.3. Calibration curve generated for Impurity 3 from 0.15 ng/mL (LOQ)
to 15 ng/mL for LOD-LOQ calculation.

Results and discussion
The LOD and LOQ were calculated using the calibration curve
method. Calibration curves from 0.15 to 15 ppb were used toestablish the LOD and LOQ.
The detection limit is expressed as DL = 3.3 × σ/S
The quantification limit is expressed as QL = 10 × σ/S
Where σ = the standard deviation of the response andS = slope of the curve.
Considering all four impurities, the LOD and LOQ are fixed as
1.0 and 3.0 ppb, respectively.

4

Figure 3.4. Calibration curve generated for Impurity 4 from 0.15 ng/mL (LOQ)
to 15 ng/mL for LOD-LOQ calculation.

Chromatographic conditions were developed to achieve
maximum separation between the impurities and API. The
API was diverted to waste to avoid severe contamination of
the MS using the integrated diverter valve. As per the diverter
valve time program, only eluent with retention times between
8 and 16 minutes proceeded to the MS.

Table 7. LOD and LOQ calculation.
Concentration in (ng/mL)
Used in Calibration Curve

Response/Area
Impurity 1

Response/Area
Impurity 2

Response/Area
Impurity 3

Response/Area
Impurity 4

0.15

585

727

1773

867

0.75

3322

4497

7527

4006

1.50

6515

9098

16017

8500

3.01

14678

18586

32129

16928

4.51

20709

27898

46782

25811

7.52

37606

48433

81777

44177

12.02

63404

80680

137752

73156

15.03

74222

97088

16042086234

Correlation

0.998760.9996

0.99880.9992STDEV (σ)

1513.71159.53195.01383.3Slope (S)5105.76592.610988.7

5887.8

LOD

0.98

0.58

0.96

0.78

LOQ

2.96

1.76

2.91

2.35

Counts

×103
6

TIC

4
2
0

Counts×103
32

Impurity 1

10Counts×103
Impurity 220Counts×103
4Impurity 3

20Counts×103
4Impurity 4

2
0
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Acquisition time (min)Figure 4. Chromatographic separation between the API and the impurity. The API is diverted to waste.

5Counts (%)×102
3

Impurity 1

2
1

Counts (%)×102
3

Impurity 2

2
1Counts (%)×102
3

Impurity 3

2
1Counts (%)×102
3

Impurity 4

2
1
9

10

11

12

13

14

15

Acquisition time (min)Figure 5. Blank chromatogram.

The reproducibility of the response is evaluated at the limit ofquantitation level by giving 10 injections of the mixture of four
impurities at a fixed LOQ level of 3 ng/mL.
Table 8. Percent RSD of 10 injections at LOQ level.
Compound NameCount

Avg. RT

Avg. Area

RSD Area

IMP 1

10

8.4

14,544.9

2.3%

IMP 2

10

10.1

18,431.6

1.4%

IMP 3

10

14.5

31,377.4

1.8%

IMP 4

10

12.3

17,824.3

1.9%

6

16

17

18

19

20

Counts

CountsCountsCountsCountsCountsCountsCountsCountsCountsTen injections at the LOQ level were performed, and the %CVof area response (impurities 1 to 4) were found to be 2.3%,
1.37%, 1.8%, and 1.9% respectively.
×103
0
×103
0
×103
0
×103
0
×103
0
×103
0
×103
0
×103
0
×103
0
×103
0
8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0

14.5

Acquisition time (min)Figure 6. Chromatogram of 10 replicate injections at the LOQ level, showing the consistency in result.

7

×104
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1

2

3

4

5

6

7

Figure 7. Matrix plot of responses of impurities 1 to 4 from 10 injections.

A linear concentration curve spanning three orders ofmagnitude was produced from 3.0 to 150 ng/mL (R2 value of0.9957 from linear regression and 1/x2 weighing). The lowest
concentration of 3 ppb ng/mL demonstrated a S/N >39:1,
63:1, 62:1, and 99:1 for impurities 1 to 4, respectively, using
the peak-to-peak algorithm for noise calculation. This
demonstrates the sensitivity of the 6470 LC/TQ, and thepossibility to analyze lower concentrations.
Table 9. Working standard preparation for plotting the calibration curve.
WorkingStandard

VolumeTaken

Volumeof Diluent

Total
VolumeResultant
Concentration

Calibration
Level

1 ppm

0.30 mL

1.7 mL

2.0 mL150 ppb

Level 81 ppm

0.20 mL

1.8 mL

2.0 mL100 ppb

Level 7150 ppb

1.0 mL

1.0 mL

2.0 mL75 ppbLevel 6100 ppb

1.0 mL

1.0 mL

2.0 mL50 ppbLevel 550 ppb

1.0 mL

1.0 mL

2.0 mL25 ppbLevel 4150 ppb

0.2 mL

1.8 mL

2.0 mL

15 ppbLevel 3100 ppb

0.2 mL

1.8 mL

2.0 mL10 ppbLevel 2150 ppb

1.0 mL

4.0 mL

5.0 mL30 ppb30 ppb

0.2 mL

1.8 mL

2.0 mL

3.0 ppb (LOQ)

8

Level 18910Preparation of spike samples

1 mL of 60/40 methanol/water was added, and vortexed
for almost two minutes. The API was partially dissolved.
However, the impurities have good solubility in the solvent
system used (60/40 methanol/water). The contents werethen filtered through PVDF filter in to a 2 mL HPLC vial for
injection. The concentrations obtained from spiked samples
were compared with the generated calibration curve.

Responses

Spiking was performed at different concentration levels,
namely LOQ, the 25 ppb level, 50 ppb level, and 75 ppb
level. 20 mg of the API was weighed in a centrifuge tube.
Appropriate volumes of working standards were spiked toachieve the desired concentration of impurities in the API.
×106
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

Impurity 1
y = 6042.724321x – 5427.314754
R2 = 0.9974

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

Concentration (ng/mL)

Figure 8. Calibration curve generated from 3 ng/mL (LOQ) to 150 ng/mL for Impurity 1.

Figure 9. Quantitative table for Impurity 1.
Table 10.1. Recovery study result for Impurity 1.
Added Impurity
Concentration

Spike ID –Impurity 1

SampleWeight

Total
Dilution

Obtained
Concentration

ActualConcentration

3 ppb

Spike 1 (LOQ)

20.0 mg

1.0 mL2.60 ppb

3.0 ppb

86.7%

25 ppb

Spike 220.0 mg

1.0 mL

25.78 ppb

25.0 ppb

103.1%

50 ppb

Spike 320.0 mg

1.0 mL

50.51 ppb

50.0 ppb

101.0%

75 ppb

Spike 420.0 mg

1.0 mL74.24 ppb

75.0 ppb

98.99%

Recovery9Responses

×106
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

Impurity 2
y = 7670.772597x – 6322.433754
R2 = 0.9983

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

Concentration (ng/mL)

Figure 10. Calibration curve generated from 3 ng/mL (LOQ) to 150 ng/mL for Impurity 2.

Figure 11. Quantitative table for Impurity 2.
Table 10.2. Recovery study result for Impurity 2.
Added Impurity
Concentration

Spike ID –Impurity 2

SampleWeight

Total
Dilution

Obtained
Concentration

ActualConcentration

Recovery

3 ppb

Spike 1 (LOQ)

20.0 mg

1.0 mL

2.78 ppb

3.0 ppb

92.7%

25 ppb

Spike 220.0 mg

1.0 mL23.22 ppb

25.0 ppb

92.9%

50 ppb

Spike 320.0 mg

1.0 mL

47.74 ppb

50.0 ppb

95.5%

75 ppb

Spike 420.0 mg

1.0 mL70.77 ppb

75.0 ppb

94.4%

10

160

Responses

×106
2.2
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

Impurity 3
y = 15077.752620x – 13035.653118
R2 = 0.9964

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

Concentration (ng/mL)

Figure 12. Calibration curve generated from 3 ng/mL (LOQ) to 150 ng/mL for Impurity 3.

Figure 13. Quantitative table for Impurity 3.
Table 10.3. Recovery study result for Impurity 3.
Added Impurity
Concentration

Spike ID –Impurity 3

SampleWeight

Total
Dilution

Obtained
Concentration

ActualConcentration

3 ppb

Spike 1 (LOQ)

20.0 mg

1.0 mL

2.73 ppb

3.0 ppb

91.0%

25 ppb

Spike 220.0 mg

1.0 mL

25.53 ppb

25.0 ppb

102.1%

50 ppb

Spike 320.0 mg

1.0 mL

52.47 ppb

50.0 ppb

104.9%

75 ppb

Spike 420.0 mg

1.0 mL80.25 ppb

75.0 ppb

107.0%

Recovery11

Responses

×106
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

Impurity 4
y = 8002.194044x – 2356.053642
R2 = 0.9953

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

Concentration (ng/mL)

Figure 14. Calibration curve generated from 3 ng/mL (LOQ) to 150 ng/mL for Impurity 4.

Figure 15. Quantitative table for Impurity 4.

Table 10.4. Recovery study result for Impurity 4.
Added Impurity
Concentration

Spike ID –Impurity 4

SampleWeight

Total
Dilution

Obtained
Concentration

ActualConcentration

Recovery

3 ppb

Spike 1 (LOQ)

20.0 mg

1.0 mL

3.19 ppb

3.0 ppb

106.3%

25 ppb

Spike 220.0 mg

1.0 mL

28.34 ppb

25.0 ppb

113.4%

50 ppb

Spike 320.0 mg

1.0 mL

57.73 ppb

50.0 ppb

115.5%

75 ppb

Spike 420.0 mg

1.0 mL

85.80 ppb

75.0 ppb

114.4%

12

RADAR plot for recovery for all impurities at four spiking levels
Impurity 1

Spike 1
Spike 1
Spike 1
Spike 1
Spike 1: at LOQ
Spike 2: at 25 ppb
Spike 3: at 50 ppb
Spike 4: at 75 ppb

Impurity 4

Impurity 2

Impurity 3
Figure 16. RADAR Plot showing the recovery percentage obtained for four different spiking levels.

Conclusion
A highly sensitive and robust MRM method was developedfor quantitation of impurities 4-chloro 2-nitrobenzene
amine, 2-chlorobenzoic acid, 2-(4-chloro 2-nitro phenyl
amino) benzoic acid, and 2-(2-amino 4-chloro phenyl amino)
benzoic acid in API, Clozapine. The chromatographic method
developed provided good separation between the impurity
and the API to avoid interference. To avoid contamination of
the MS, an integrated diverter valve program was included
to divert high concentration API as it elutes. The method
showed an LOD of 1 ppb and LOQ of 3 ppb, calculated by
the calibration curve method. Calibration curves were madebetween LOQ (3 ppb) level to 150 ppb for all four impurities,
and were found to be linear with 1/x2 weighing.

The minimum signal to noise ratios for impurities 1 to 4 at
the LOQ level were found to be 39:1, 63:1, 62:1, and 99:1
respectively. R2 values were above 0.9950. Spike recovery
analysis showed the efficiency of sample extraction
with recovery percentage between 85 to 116%, at a test
concentration of 20 mg/mL. The method developed wasfound to be highly reproducible at the LOQ level, with %RSD
value for the area response of 10 replicate injections not morethan 2.5%. The method can be used for quality control of the
clozapine API.

13

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www.agilent.com
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This information is subject to change without notice.
© Agilent Technologies, Inc. 2022
Published in the USA, July 8, 2022
5994-4991EN