Page 28 - Clinical-Medical Solution for Clinical Chemistry

Application AD-0135
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 Results and Discussion (x1,000,000)
1.00
MER
Fast MRM-based method for five β-lactam antibiotics 0.75
Table 2 shows the summarized results of optimized MRM
transitions and parameters of the five β-lactam antibiotics 0.50
studied and four stable isotope-labelled internal standards. 0.25 MER-d6
Two MRM transitions were selected for each compound, with
one as the quantitation ion and the other for confirmation. 0.00
(x1
0.000,000) 1.0 2.0 3.0 min
Furthermore, a fast gradient elution MRM method was MER-d6
established with a total run time of 5 minutes. The MRM 2.0 TAZ
chromatograms of a mixed standard sample in plasma are
shown in Figure 3. Due to lack of stable isotope-labelled 1.0
tazobactam, MER-d6 was also used as the internal standard
for tazobactam (TAZ) in this work. 0.0
0.0 1.0 2.0 3.0 min
(x100,000)
Table 2: MRM transitions and parameters of five β-lactam antibiotics 2.0 CEF
and internal standards on LCMS-8060
1.5
Compd. Formula E. Mass MRM (m/z) CE (V) Int (%)
1.0
301.1>168.2 -15 100 CEF-cd3
Tazobactam C 10H 12N 4O 5 300.05
(TAZ) S 301.1>122.1 -22 92 0.5
481.1>86.2 -15 100 0.0
Cefepime C 19H 24N 6O 5 480.13
(x100,000)
(CEF) S 2 481.1>396.0 -13 63 0.0 1.0 2.0 3.0 min
1.5
384.1>68.1 -41 100
Meropenem C 17H 25N 3O 5 383.15
(MER) S 1.0 CFT-d6 CFT
384.1>141.1 -16 64
547.1>468.0 -13 100 0.5
Ceftazidime C 22H 22N 6O 7 546.10
(CFT) S 2 547.1>396.1 -19 42
0.0
518.2>143.1 -21 100
(x1,000,000)
Piperacillin C 23H 27N 5O 7 517.16 0.0 1.0 2.0 3.0 min
(PIP) S 518.2>160.1 -15 25 1.00 PIP
13
C 18 CH 21D 485.2>86.1 -16 100 0.75
CEF-cd3 484.13 PIP-d5
3N 6O 5S 2 485.2>400.1 -13 66 0.50
390.2>147.2 -18 100
MER-d6 C 17H 19D 6N 3 389.15 0.25
O 5S 390.2>114.1 -27 74
0.00
553.1>474.0 -16 100
CFT-d6 C 22H 16D 6N 3 552.10 0.0 1.0 2.0 3.0 min
O 7S 2 553.1>319.1 -20 59 Figure 3: MRM chromatograms of five β-lactam antibiotics each (400
523.1>148.1 -21 100 ng/mL) with internal standards in plasma on LCMS-8060
PIP-d5 C 23H 22D 5N 5 522.16
O 7S 523.1>160.1 -14 23 R2 greater than 0.997 was obtained for the five compounds in
the range from 20 ng/mL to 4000 ng/mL in plasma.
Table 3: MRM-based quantitation method of five β-lactam antibiotics Evaluation of method performance
with internal standards on LCMS-8060
Accuracy of the quantitation method was evaluated with pre-
RT IS Range spiked standard samples at all concentration levels with
Compd. qMRM (m/z) IS R 2
(min) (ng/mL) (ng/mL) duplicate injections. The results are shown in Table 4, which
TAZ 1.18 301.1 > 168.1 MER-d6 200 20~4000 0.9993 indicate that reliable quantitation accuracy was obtained,
CEF 1.21 481.1 > 86.1 CEF-cd3 800 20~4000 0.9991 except CFT at 20 ng/mL, due to employing IS method.
MER 1.47 384.1 > 68.1 MER-d6 200 20~4000 0.9989 Table 4: Results of RSD (%, n=5) of five β-lactam antibiotics with IS
in plasma samples on LCMS-8060
CFT 1.47 547.1 > 468.0 CFT-d6 800 20~4000 0.9971
Accuracy (%)
PIP 2.33 518.2 > 143.1 PIP-d5 200 20~4000 0.9999
Compd. 20 40 80 200 400 2000 4000
Calibration curves with IS ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL
TAZ 91 97 100 106 107 101 99
As shown in Figure 4, linear calibration curves with IS method CEF 87 97 102 105 109 101 98
were constructed using the standard samples prepared by MER 97 102 102 99 102 95 102
pre-spiked in plasma matrix. The method parameters are CFT 126 103 93 87 91 96 104
summarized in Table 3. It can be seen that good linearity with PIP 99 100 97 103 102 100 100