Table 3. Accuracy, Precision and...
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Table 1. Timed SIM Scan Events Analyte t R (min) SIM (m/z) Timed Event (min) Scan Time (s) Benzoate 3.9 121 3.6–5.2 0.2 p-Hydroxybenzoate 4.4 137 3.6–5.2 0.2 Citrate 5.8 191 5.2–10.0 0.2 IS (citrate-d 4 ) 5.8 195 5.2–10.0 0.2 Etidronate 6.6 205 5.2–10.0 0.4 Clodronate 7.3 243 5.2–10.0 0.4 Tiludronate 12.1 317 10.0–14.0 1.0 Table 2. Calibration and Range, Precision, and MDL Analyte Calibration Range r 2 Fit %RSD a (n=5) MDL b Benzoate 5–500 0.9994 Quadratic 8.00 2.48 p-Hydroxybenzoate 5–500 0.9998 Quadratic 5.31 1.20 Citrate 5–500 0.9997 Linear 3.82 1.31 Etidronate 50–500 0.9978 Quadratic 5.33 9.36 Clodronate 50–500 0.9970 Quadratic 10.28 15.5 Tiludronate 50–500 0.9957 Quadratic 4.51 7.19 a %RSD was calculated based on 20 pg injection for benzoate, p-hydroxybenzoate and citrate; and 100 pg injection for bisphosphonates; b MDL was calculated by MDL = S × t 99%, n=5 where S is the standard deviation and t is the Student’s t at 99% confidence interval. Table 3. Accuracy, Precision and Recovery Analyte 50 ppb (n=3) 200 ppb (n=3) Original Observed % Recovery * Mean % RSD % Accuracy Mean % RSD % Accuracy Benzoate 54.2 2.75 108 499 4.71 99.9 ND 89.5 89.5 p-Hydroxybenzoate 52.4 1.53 105 499 7.07 99.9 ND 93.5 93.5 Citrate 49.4 2.58 98.7 497 0.76 99.5 ND 102 102 Etidronate 43.4 4.27 86.8 498 2.18 99.5 424 542 117 Clodronate 44.8 4.30 89.7 498 1.05 99.6 ND 134 134 Tiludronate 41.5 3.26 83.0 497 1.28 99.4 ND 121 121 Unit shown in this table: ppb. *Recovery was calculated based on [observed amount (original sample+100 ppb spiked each analyte) – original amount]/100 × 100% 29053 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 4 Suppressed Conductivity μS Minutes Concentration: 40.00 mM 50.00 100.00 40.00 F Cl NO 2 HSO 4 Br NO 3 H 2 PO 4 Benzoate OH-benzoate Citrate Etidronate Clodronate Tiludronate System: Dionex ICS-5000 RFIC Columns: Dionex IonPac AS18-Fast (0.4 mm) Temperature: 40 °C Eluent Source: EGC-KOH Capillary Cartridge Eluent: Hydroxide Gradient Time (min) OH Conc -4.0 40 mM 5.0 50 mM 8.0 100 mM 14.9 100 mM 15.0 40 mM Flow Rate: 20 μL/min Injection: 10 μL, 20–100 ppb each 29054 Clodronate 0 108 50 100 150 200 250 300 350 400 450 500 243.0 143.0 78.9 488.6 191.1 264.9 121.1 170.9 % [M-H] - C P P Cl Cl O O 0 108 240 242 244 246 248 250 243.0 245.0 247.0 % m/z 0 108 314.9 318.0 320.0 322.0 325.1 316.9 318.9 318.0 319.9 320.9 % m/z 0 108 50 100 150 200 250 300 350 400 450 500 205.1 122.9 191.1 411.0 432.8 227.1 80.9 102.0 143.1 272.8 62.9 454.8 169.0 299.2 % [M-H] - C P P CH 3 O OH OH OH OH OH OH OH O HO HO Etidronate [M-H-H 3 PO 3 ] - [M-H-HPO 3 ] - Tiludronate 0 108 50 100 150 200 250 300 350 400 450 500 316.9 237.1 78.9 178.7 298.9 158.0 338.9 106.1 199.1 % C P P H S O OH OH OH O HO CI [M-H] - CH 4 35 Cl 2 P 2 O 6 CH 4 35 Cl 37 ClP 2 O 6 CH 4 37 Cl 2 P 2 O 6 C 7 H 9 35 Cl 32 SP 2 O 6 C 7 H 9 35 Cl 34 SP 2 O 6 + C 7 H 9 37 Cl 32 SP 2 O 6 C 7 H 9 37 Cl 34 SP 2 O 6 29055 4.5e3 Chromatographic Conditions System: Dionex ICS-5000 RFIC Columns: Dionex IonPac AS18-Fast (0.4 mm) Temperature: 40 °C Mobile Phase: Hydroxide from Dionex EGC-KOH Capillary Cartridge Time (min) OH Conc -4.0 40mM 5.0 50 mM 8.0 100 mM 14.9 100 mM 15.0 40 mM Flow Rate: 20 μL/min Injection: 0.2 ng in 2 μL MS Conditions System: Thermo Scientific MSQ Plus Mass Spectrometric Detector Interface: Electrospray ionization (ESI) Probe Temp: 300 °C Needle Voltage: 3500 V Solvent: 20 μL/min acetonitrile Nebulizer Gas: Nitrogen at 65 psi Detection Mode: Selected ion monitoring (SIM) Tiludronate SIM_12: 317 m/z 2.4e3 Clodronate SIM_11: 243 m/z 4.5e3 Etidronate SIM_10: 205 m/z 1.4e4 Citrate SIM_09: 191 m/z 1.6e4 SIM_08:137 m/z p-OH-benzoate Counts 0 2 4 6 8 10 12 14 0 2.2e4 SIM_07: 121 m/z Minutes Benzoate
Transcript of Table 3. Accuracy, Precision and...
Table 1. Timed SIM Scan EventsAnalyte tR (min) SIM (m/z) Timed Event (min) Scan Time (s)
Benzoate 3.9 121 3.6–5.2 0.2p-Hydroxybenzoate 4.4 137 3.6–5.2 0.2Citrate 5.8 191 5.2–10.0 0.2IS (citrate-d4) 5.8 195 5.2–10.0 0.2Etidronate 6.6 205 5.2–10.0 0.4Clodronate 7.3 243 5.2–10.0 0.4Tiludronate 12.1 317 10.0–14.0 1.0
Table 2. Calibration and Range, Precision, and MDL
Analyte Calibration Range r2 Fit %RSDa (n=5) MDLb
Benzoate 5–500 0.9994 Quadratic 8.00 2.48p-Hydroxybenzoate 5–500 0.9998 Quadratic 5.31 1.20Citrate 5–500 0.9997 Linear 3.82 1.31Etidronate 50–500 0.9978 Quadratic 5.33 9.36Clodronate 50–500 0.9970 Quadratic 10.28 15.5Tiludronate 50–500 0.9957 Quadratic 4.51 7.19
a %RSD was calculated based on 20 pg injection for benzoate, p-hydroxybenzoate and citrate; and 100 pg injection for bisphosphonates;b MDL was calculated by MDL = S × t99%, n=5 where S is the standard deviation and t is the Student’s t at 99% confi dence interval.
Table 3. Accuracy, Precision and Recovery
Analyte50 ppb (n=3) 200 ppb (n=3)
Original Observed %Recovery*Mean %
RSD%
Accuracy Mean %RSD
%Accuracy
Benzoate 54.2 2.75 108 499 4.71 99.9 ND 89.5 89.5p-Hydroxybenzoate 52.4 1.53 105 499 7.07 99.9 ND 93.5 93.5Citrate 49.4 2.58 98.7 497 0.76 99.5 ND 102 102Etidronate 43.4 4.27 86.8 498 2.18 99.5 424 542 117Clodronate 44.8 4.30 89.7 498 1.05 99.6 ND 134 134Tiludronate 41.5 3.26 83.0 497 1.28 99.4 ND 121 121
Unit shown in this table: ppb.*Recovery was calculated based on [observed amount (original sample+100 ppb spiked each analyte) – original amount]/100 × 100%
290530 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
4Suppressed Conductivity
µS
Minutes
Concentration: 40.00 mM
50.00
100.00
40.00
F
Cl
NO2HSO4
Br NO3
H2PO4
BenzoateOH-benzoateCitrate
EtidronateClodronate
Tiludronate
System: Dionex ICS-5000 RFICColumns: Dionex IonPac AS18-Fast (0.4 mm)Temperature: 40 °CEluent Source: EGC-KOH Capillary CartridgeEluent: Hydroxide Gradient Time (min) OH Conc -4.0 40 mM 5.0 50 mM 8.0 100 mM 14.9 100 mM 15.0 40 mM
Flow Rate: 20 µL/minInjection: 10 µL, 20–100 ppb each
29054
Clodronate
0
108
50 100 150 200 250 300 350 400 450 500
243.0
143.078.9488.6191.1 264.9121.1 170.9
%
[M-H]-
CP P
Cl
Cl OO
0
108
240 242 244 246 248 250
243.0
245.0
247.0
%
m/z
0
108
314.9 318.0 320.0 322.0 325.1
316.9
318.9
318.0319.9 320.9
%
m/z
0
108
50 100 150 200 250 300 350 400 450 500
205.1
122.9
191.1 411.0 432.8227.180.9 102.0 143.1 272.862.9 454.8169.0 299.2
%
[M-H]-
CP P
CH3 O
OH
OH
OH OH OH
OH OH
O
HO
HO
Etidronate
[M-H-H3PO3]-
[M-H-HPO3]-
Tiludronate
0
108
50 100 150 200 250 300 350 400 450 500
316.9
237.1
78.9 178.7 298.9158.0 338.9106.1 199.1
%CP P
H
S O
OH
OH OH
O
HO
CI[M-H]-
CH435Cl2P2O6
CH435Cl37ClP2O6
CH437Cl2P2O6
C7H935Cl32SP2O6
C7H935Cl34SP2O6+
C7H937Cl32SP2O6
C7H937Cl34SP2O6
29055
4.5e3 Chromatographic ConditionsSystem: Dionex ICS-5000 RFICColumns: Dionex IonPac AS18-Fast (0.4 mm)Temperature: 40 °CMobile Phase: Hydroxide from Dionex EGC-KOH Capillary Cartridge Time (min) OH Conc -4.0 40mM 5.0 50 mM 8.0 100 mM 14.9 100 mM 15.0 40 mMFlow Rate: 20 µL/minInjection: 0.2 ng in 2 µL
MS ConditionsSystem: Thermo Scientific MSQ Plus Mass Spectrometric DetectorInterface: Electrospray ionization (ESI)Probe Temp: 300 °CNeedle Voltage: 3500 VSolvent: 20 µL/min acetonitrileNebulizer Gas: Nitrogen at 65 psiDetection Mode: Selected ion monitoring (SIM)
TiludronateSIM_12: 317 m/z
2.4e3 Clodronate SIM_11: 243 m/z
4.5e3 Etidronate SIM_10: 205 m/z
1.4e4 Citrate SIM_09: 191 m/z
1.6e4 SIM_08:137 m/z p-OH-benzoate
Cou
nts
0 2 4 6 8 10 12 140
2.2e4 SIM_07: 121 m/z
Minutes
Benzoate
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