Quantitative analysis of Mometasone on LCMS-8060

Confidential

Sample details

➢ Extracted samples for 2 PA batches and post spiked samples (LQC,MQC & HQC)

Sr.No Batch Sample type Sample details Total no.

Blank, zero standard, 1 set

1 PA_BATCH_01 Wet extract CC and 3 set QCs (LLQC, 23
LQC, MQC & HQC)
Blank, zero standard, 1 set
CC and 3 set QCs (LLQC,
2 PA_BATCH_02 Dry extract 23
LQC, MQC & HQC)

3 sets of QCs (LQC,MQC &

3 Recovery samples Post spiked 6
HQC)

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Batch organization details

➢ PA_BATCH_01 followed by post spike recovery samples were analyzed as

such.

➢ PA_BATCH_02 samples were reconstituted and analyzed.

➢ PA_BATCH_01 was reinjected as PA_BATCH_01R.

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Mometasone Results

Batch Linearity range LLOQ area LLOQ Correlation

(pg/mL) counts (S/N) coefficient (r)
PA_BATCH_01 2422 149.19 0.9985
0.255 to 29.971
PA_BATCH_01R 2458 59.66 0.9982
PA_BATCH_02 2954 119.63 0.9953

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Analytical Conditions for Mometasone
-LC-
Nexera
Column Kinetex 2.6 µm C8 100 A, 150×4.6 mm
Mobile Phase Acetonitrile : 2 mM Ammonium trifluoroacetate
Flow Rate Flow gradient

Oven Temperature 40 deg. C

Injection Volume 40 uL

Note: Acetonitrile used was Lichrosolve from Merck. As per Customer use of acetonitrile from Biosolve increases the sensitivity and reduces
noise which can be an additional factor to improve chromatography still.

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Analytical Conditions for Mometasone
-MS-
LCMS-8060

Interface Electro Spray Ionization (ESI)

Polarity Positive

MRM Transitions Mometasone (521.20 > 355.05)

Mometasone D3 (524.20 > 355.20)
Nebulizing Gas Flow 3.0 L / min

Heating gas and Drying Gas 18 L/min and 5 L / min

Flow
Interface Temp. 300 deg. C

DL and Heat block Temp. 150 and 400 deg. C

Interface Voltage 0.5 KV

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Summary of Result – Calibration curve

Calibration Standard PA_BATCH_01 PA_BATCH_02 PA_BATCH_01R

standards concentration
Accuracies (%)
(pg/mL)
CS_09 29.971 103.23 100.94 102.73
CS_08 24.127 104.7 105.07 101.84
CS_07 18.095 103.6 101.09 104.77
CS_06 12.088 97.24 101.87 99.93
CS_05 6.044 100.66 100.8 103.69
CS_04 3.022 99.74 100.06 97.49
CS_03 1.511 89.06 88.37 93.33
CS_02 0.51 45.02 77.19 90.28
CS_01 0.255 101.77 101.81 105.93

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Representative extracted blank chromatogram

Q 521.20>355.05 (+) 1.75e3

1.6e3

1.4e3

1.2e3

1.0e3

8.0e2

6.0e2

4.0e2

2.0e2

0.0e0
0 1 2 3 4 5 6 7 8 9
RT (min)

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Representative extracted LLOQ chromatogram

Q 521.20>355.05 (+)

2.2e3
2.0e3
1.8e3
1.6e3
1.4e3
1.2e3
1.0e3
8.0e2 RT=6.168
6.0e2
4.0e2
2.0e2
0.0e0
0 1 2 3 4 5 6 7 8 9
RT (min)

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Representative calibration curve

Area Ratio
MOMETASONE
y = 0.06053453x + 0.01685852
1.8 R² = 0.9964226 R = 0.9982097
Curve Fit: Default (Linear)
Weighting: Default (1/C^2)
1.6
Zero: Default (Not Forced)
Mean RF: 7.412124e-002
1.4 SD RF: 2.250709e-002
%RSD: 30.365229
1.2

1.0

0.8

0.6

0.4

0.2

0.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Conc.Ratio (pg/mL)

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Conclusion

• The sensitivity for mometasone was achieved with LLOQ of 0.2 pg/mL in
extracted plasma samples using Shimadzu LCMS-8060.

• The chromatography is good and consistent in all the 3 batches with S/N
for 0.2 pg/mL well above 50.

• The accuracy failures at lower levels might be due to internal standard

area variation.

• The reinjected batch (PA_BATCH_01R) shows better results in terms of

increased area counts and good accuracies after the system stabilization.

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ID1: MOMETASONE
Correlation coefficient: R² = 0.9964226 R = 0.9982097
Calibration curve: Curve type: Linear | Zero: Not Forced | Weighting: 1/C^2 | Equation: y = 0.06053453x + 0.01685852
Processing Method: Internal Standard
Area Ratio

1.5

1.0

0.5

0.0
0 10 20 30
Conc.Ratio (pg/mL)

Sample Name Sample Type Area ISTD Area Area Ratio S/N Std. Conc. Conc. Level Cal Point Accuracy Acquired Date Data Filename Inj Vol
(%)
BLK Unknown 730 228 3.20 41.38 ---- 52.61 ---- ---- ---- 13-06-2019 22:52:25 130619_PA_R_001 40.0000
ZS Unknown 96 89060 0.00 6.59 ---- -0.2607 ---- ---- ---- 13-06-2019 23:02:46 130619_PA_R_002 40.0000
CS_09 Standard 185654 98713 1.88 8691.84 29.971 30.79 9 Yes 102.73 13-06-2019 23:13:11 130619_PA_R_003 40.0000
CS_08 Standard 143377 95318 1.50 4373.06 24.127 24.57 8 Yes 101.84 13-06-2019 23:23:31 130619_PA_R_004 40.0000
CS_07 Standard 113948 97851 1.16 4329.97 18.095 18.96 7 Yes 104.77 13-06-2019 23:33:55 130619_PA_R_005 40.0000
CS_06 Standard 73709 98527 0.75 3361.10 12.088 12.08 6 Yes 99.93 13-06-2019 23:44:17 130619_PA_R_006 40.0000
CS_05 Standard 37017 93420 0.40 894.36 6.044 6.267 5 Yes 103.69 13-06-2019 23:54:37 130619_PA_R_007 40.0000
CS_04 Standard 19144 98076 0.20 496.60 3.022 2.946 4 Yes 97.49 14-06-2019 00:05:00 130619_PA_R_008 40.0000
CS_03 Standard 10446 102184 0.10 353.86 1.511 1.410 3 Yes 93.33 14-06-2019 00:15:21 130619_PA_R_009 40.0000
CS_02 Standard 4238 94747 0.04 71.14 0.510 0.4604 2 Yes 90.28 14-06-2019 00:25:43 130619_PA_R_010 40.0000
CS_01 Standard 2458 74012 0.03 59.66 0.255 0.2701 1 Yes 105.93 14-06-2019 00:36:03 130619_PA_R_011 40.0000
HQC_01 Control 110349 76481 1.44 2497.45 23.686 23.56 13 ---- 99.45 14-06-2019 00:46:24 130619_PA_R_012 40.0000
MQC_01 Control 79061 105711 0.75 1586.20 12.696 12.08 12 ---- 95.12 14-06-2019 00:56:48 130619_PA_R_013 40.0000
LQC_01 Control 8333 155459 0.05 371.62 0.696 0.6070 11 ---- 87.21 14-06-2019 01:07:13 130619_PA_R_014 40.0000
LLQC_01 Control 3177 96582 0.03 91.73 0.255 0.2649 10 ---- 103.88 14-06-2019 01:17:37 130619_PA_R_015 40.0000
HQC_02 Control 116839 83023 1.41 3615.38 23.686 22.97 13 ---- 96.98 14-06-2019 01:27:57 130619_PA_R_016 40.0000
MQC_02 Control 78162 104022 0.75 1521.24 12.696 12.13 12 ---- 95.58 14-06-2019 01:38:17 130619_PA_R_017 40.0000
LQC_02 Control 4441 103568 0.04 124.30 0.696 0.4299 11 ---- 61.76 14-06-2019 01:48:41 130619_PA_R_018 40.0000
LLQC_02 Control 3077 100334 0.03 65.02 0.255 0.2281 10 ---- 89.46 14-06-2019 01:59:04 130619_PA_R_019 40.0000
HQC_03 Control 145207 102676 1.41 3699.76 23.686 23.08 13 ---- 97.46 14-06-2019 02:09:27 130619_PA_R_020 40.0000
MQC_03 Control 84142 111014 0.76 941.64 12.696 12.24 12 ---- 96.43 14-06-2019 02:19:55 130619_PA_R_021 40.0000
LQC_03 Control 4950 99207 0.05 145.81 0.696 0.5458 11 ---- 78.41 14-06-2019 02:30:18 130619_PA_R_022 40.0000
LLQC_03 Control 2512 99933 0.03 75.71 0.255 0.1368 10 ---- 53.63 14-06-2019 02:40:40 130619_PA_R_023 40.0000

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BLK BLK ZS ZS
Conc 52.61 Conc 1.000 Conc -0.2607 Conc 1.000
Area 730 Area 228 Area 96 Area 89060
Height 140 Height 30 Height 30 Height 11804
Q 521.20>355.05 (+) 1.42e3 ISTD 524.20>355.20 (+) 3.81e2 Q 521.20>355.05 (+) 7.25e3 ISTD 524.20>355.20 (+) RT=6.168 1.19e4

1.3e3
6.0e3 1.0e4
3.0e2
1.0e3
7.5e3
7.5e2 2.0e2 4.0e3
RT=6.207 5.0e3
RT=6.163
5.0e2
1.0e2 2.0e3
2.5e3
2.5e2
RT=6.199

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

CS_09 CS_09 CS_08 CS_08

Conc 30.79 Conc 1.000 Conc 24.57 Conc 1.000
Area 185654 Area 98713 Area 143377 Area 95318
Height 30691 Height 13115 Height 23742 Height 12661
Q 521.20>355.05 (+) 3.09e4 ISTD 524.20>355.20 (+) 1.32e4 Q 521.20>355.05 (+) 2.40e4 ISTD 524.20>355.20 (+) 1.28e4
RT=6.183 RT=6.169 RT=6.183 RT=6.169
3.0e4 1.3e4 1.3e4

2.0e4
1.0e4 1.0e4

2.0e4 1.5e4
7.5e3 7.5e3

1.0e4 5.0e3
5.0e3
1.0e4

2.5e3 5.0e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

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CS_07 CS_07 CS_06 CS_06
Conc 18.96 Conc 1.000 Conc 12.08 Conc 1.000
Area 113948 Area 97851 Area 73709 Area 98527
Height 18883 Height 12957 Height 12134 Height 13062
Q 521.20>355.05 (+) 1.91e4 ISTD 524.20>355.20 (+) 1.31e4 Q 521.20>355.05 (+) 1.24e4 ISTD 524.20>355.20 (+) 1.32e4
RT=6.183 RT=6.170 RT=6.183 RT=6.169

1.3e4 1.3e4

1.5e4 1.0e4
1.0e4 1.0e4

7.5e3
7.5e3 7.5e3
1.0e4

5.0e3 5.0e3 5.0e3

5.0e3
2.5e3 2.5e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

CS_05 CS_05 CS_04 CS_04

Conc 6.267 Conc 1.000 Conc 2.946 Conc 1.000
Area 37017 Area 93420 Area 19144 Area 98076
Height 6155 Height 12402 Height 3069 Height 12935
Q 521.20>355.05 (+) 6.38e3 ISTD 524.20>355.20 (+) 1.25e4 Q 521.20>355.05 (+) 3.29e3 ISTD 524.20>355.20 (+) 1.31e4
RT=6.180 RT=6.165 RT=6.186 RT=6.171
1.3e4
6.0e3 1.3e4
3.0e3
1.0e4 1.0e4

4.0e3 7.5e3 2.0e3

7.5e3

5.0e3 5.0e3
2.0e3 1.0e3
2.5e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

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CS_03 CS_03 CS_02 CS_02
Conc 1.410 Conc 1.000 Conc 0.4604 Conc 1.000
Area 10446 Area 102184 Area 4238 Area 94747
Height 1704 Height 13485 Height 625 Height 12512
Q 521.20>355.05 (+) 2.12e3 ISTD 524.20>355.20 (+) 1.36e4 Q 521.20>355.05 (+) 2.49e3 ISTD 524.20>355.20 (+) 1.26e4
RT=6.169 RT=6.169
RT=6.185 1.3e4
2.0e3 1.3e4
2.0e3 1.0e4
1.5e3 1.0e4
1.5e3 7.5e3
7.5e3
1.0e3 RT=6.185
1.0e3 5.0e3
5.0e3
5.0e2
2.5e3 5.0e2 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

CS_01 CS_01 HQC_01 HQC_01

Conc 0.2701 Conc 1.000 Conc 23.56 Conc 1.000
Area 2458 Area 74012 Area 110349 Area 76481
Height 354 Height 9885 Height 18411 Height 10218
Q 521.20>355.05 (+) 3.14e3 ISTD 524.20>355.20 (+) 1.00e4 Q 521.20>355.05 (+) 1.87e4 ISTD 524.20>355.20 (+) 1.04e4
RT=6.173 RT=6.184 RT=6.171
1.0e4
3.0e3 1.0e4

8.0e3 1.5e4 8.0e3

2.0e3
6.0e3 6.0e3
1.0e4

4.0e3 4.0e3
1.0e3
RT=6.189 5.0e3
2.0e3 2.0e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

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MQC_01 MQC_01 LQC_01 LQC_01
Conc 12.08 Conc 1.000 Conc 0.6070 Conc 1.000
Area 79061 Area 105711 Area 8333 Area 155459
Height 12967 Height 14006 Height 1323 Height 20601
Q 521.20>355.05 (+) 1.33e4 ISTD 524.20>355.20 (+) 1.42e4 Q 521.20>355.05 (+) 2.92e3 ISTD 524.20>355.20 (+) 2.08e4
RT=6.188 RT=6.174 RT=6.174

1.3e4 2.0e4
1.3e4
1.0e4 1.5e4
1.0e4 2.0e3 RT=6.186
7.5e3
7.5e3
1.0e4
5.0e3 5.0e3 1.0e3
5.0e3
2.5e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

LLQC_01 LLQC_01 HQC_02 HQC_02

Conc 0.2649 Conc 1.000 Conc 22.97 Conc 1.000
Area 3177 Area 96582 Area 116839 Area 83023
Height 511 Height 12814 Height 19729 Height 11104
Q 521.20>355.05 (+) 1.93e3 ISTD 524.20>355.20 (+) 1.30e4 Q 521.20>355.05 (+) 2.00e4 ISTD 524.20>355.20 (+) 1.12e4
RT=6.170 RT=6.185 RT=6.172
2.0e4
1.3e4
1.0e4
1.5e3 1.0e4 1.5e4
8.0e3
7.5e3
1.0e3 RT=6.184 6.0e3
1.0e4
5.0e3 4.0e3
5.0e2 5.0e3
2.5e3 2.0e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

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MQC_02 MQC_02 LQC_02 LQC_02
Conc 12.13 Conc 1.000 Conc 0.4299 Conc 1.000
Area 78162 Area 104022 Area 4441 Area 103568
Height 12957 Height 13795 Height 742 Height 13745
Q 521.20>355.05 (+) 1.33e4 ISTD 524.20>355.20 (+) 1.40e4 Q 521.20>355.05 (+) 1.89e3 ISTD 524.20>355.20 (+) 1.39e4
RT=6.190 RT=6.175 RT=6.171

1.3e4
1.3e4 1.3e4
1.5e3
1.0e4
1.0e4 1.0e4
RT=6.187
7.5e3 7.5e3 7.5e3
1.0e3

5.0e3 5.0e3 5.0e3

5.0e2
2.5e3 2.5e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

LLQC_02 LLQC_02 HQC_03 HQC_03

Conc 0.2281 Conc 1.000 Conc 23.08 Conc 1.000
Area 3077 Area 100334 Area 145207 Area 102676
Height 471 Height 13357 Height 23980 Height 13694
Q 521.20>355.05 (+) 1.69e3 ISTD 524.20>355.20 (+) 1.35e4 Q 521.20>355.05 (+) 2.42e4 ISTD 524.20>355.20 (+) 1.39e4
RT=6.171 RT=6.185 RT=6.172

1.3e4 1.3e4
1.5e3
2.0e4
1.0e4 1.0e4
1.0e3 1.5e4
RT=6.185 7.5e3 7.5e3
1.0e4
5.0e3 5.0e3
5.0e2
2.5e3 5.0e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

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MQC_03 MQC_03 LQC_03 LQC_03
Conc 12.24 Conc 1.000 Conc 0.5458 Conc 1.000
Area 84142 Area 111014 Area 4950 Area 99207
Height 13922 Height 14644 Height 763 Height 13148
Q 521.20>355.05 (+) 1.42e4 ISTD 524.20>355.20 (+) 1.48e4 Q 521.20>355.05 (+) 1.91e3 ISTD 524.20>355.20 (+) 1.33e4
RT=6.188 RT=6.173 RT=6.169

1.3e4
1.3e4
1.5e3
1.0e4
1.0e4 1.0e4 RT=6.184
7.5e3
7.5e3 1.0e3

5.0e3 5.0e3
5.0e3
5.0e2
2.5e3 2.5e3

0.0e0 0.0e0 0.0e0 0.0e0

0 2 4 6 8 2 4 6 8 0 2 4 6 8 2 4 6 8
RT (min) RT (min) RT (min) RT (min)

LLQC_03 LLQC_03
Conc 0.1368 Conc 1.000
Area 2512 Area 99933
Height 436 Height 13283
Q 521.20>355.05 (+) 2.85e3 ISTD 524.20>355.20 (+) 1.35e4
RT=6.173

1.3e4
2.5e3

1.0e4
2.0e3

1.5e3 7.5e3

1.0e3 RT=6.189 5.0e3

5.0e2 2.5e3

0.0e0 0.0e0
0 2 4 6 8 2 4 6 8
RT (min) RT (min)

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