44 / 658
2
Analytical separation was carried out on a
Thermo Scientific Hypercarb column (50×3 mm,
5.0-µm particle size) at 70 °C. The total LC runtime
was 12 minutes (Figure 1). The mass spectrometer was
operated with a heated electrospray ionization (HESI-II)
source in positive ionization mode. Data was acquired in
selected-reaction monitoring (SRM) mode.
Validation
The validation procedure included tests for 1) recovery;
2) lower limit of quantitation (LLOQ), dynamic range,
accuracy; 3) precision; 4) ion suppression; 5) carryover;
and 6) interferences.
Results and Discussion
Charcoal stripped serum (CSS) was first evaluated by
comparing it to human plasma using a generally adopted
mixing study
3
. It was determined that CSS is an
appropriate matrix to conduct the validation experiments.
Recovery
The extraction recovery was assessed by comparing the
direct injection to the TurboFlow method injection of MN,
NMN, MN-d3 and NMN-d3 spiked in mobile phase (n=2).
The absolute recovery of MN, NMN and their IS ranged
from 56.4% to 62.4%, and the relative recovery of MN and
NMN was 90.9% and 97.8%, respectively (Table 1).
Table 1. Recovery
Determination of LLOQ, Linearity and Accuracy
CSS was spiked with MN and NMN to achieve final
concentrations of 500 and 1000 pg/mL, respectively. A
serial two-fold dilution with CSS was performed to make
eight levels of linearity samples with concentration ranges
of 500 to 3.9 pg/mL and 1000 to 7.8 pg/mL for MN and
NMN, respectively. Linearity samples were analyzed in
triplicate along with one set of calibrators. The calibration
curve was constructed by plotting the
analyte:ISpeak area
ratio vs. analyte concentration.
The linearity was determined to be 6.3 to 455.4 pg/mL for
MN and 12.6 to 954.5 pg/mL for NMN. Within the linear
range, the accuracy ranged from 80.6% to 93.5% for
MN, and from 80.9% to 101.7% for NMN. The CV
(n=3) from all linearity levels ranged from 3.1% to
13.7% for MN, and from 1.6% to 10.7% for NMN
(Table 1 and Figures 2 and 3). The determined LLOQ was
6.3 pg/mL for MN and 12.6 pg/mL for NMN (Table 2).
Online
Extraction
(mean ± CV)
b
Direct
Injection
(mean ± CV)
Absolute
Recovery
(%)
Relative
Recovery
(%)
MN (500 pg/mL)
a
NMN (250 pg/mL)
a
60281 ± 2.7%
32186 ± 5.6%
106866 ± 10.5%
51878 ± 9.4%
56.4
62.0
90.9
97.8
MN-d3 (500 pg/mL)
a
NMN-d3 (500 pg/mL)
a
40716 ± 1.1%
28983 ± 3.7%
66790 ± 11.4%
46482 ± 11.8%
61.0
62.4
N/A
N/A
a
MN, NMN, MN-d3 and NMN-d3 were spiked to mobile phase
at specified concentration levels.
b
Measured peak area with CV (n=2)
MN
NMN
Dilution
factor
Expected
(pg/mL)
Measured
(pg/mL)
CV of
triplicates (%)
Accuracy
(%)
Expected
(pg/mL)
Measured
(pg/mL)
CV of
triplicates (%)
Accuracy
(%)
128
3.91
5.5
17.2
71.1
7.8
7.4
35.3
94.9
64
7.81
6.3
13.7
80.6
15.6
12.6
10.7
80.9
32
15.6
13.9
7.2
88.8
31.3
30.8
1.6
98.7
16
31.3
27.5
4.9
88.0
62.5
61.0
6.0
98.1
8
62.5
56.6
10.3
90.6
125.0
121.2
9.2
96.9
4
125.0
112.2
4.0
89.8
250.0
254.2
9.4
101.7
2
250.0
233.7
3.1
93.5
500.0
496.9
2.7
99.4
1
500.0
455.4
4.0
91.1
1000.0
954.5
3.3
95.5
Mean (%)
88.9
95.9
Stdev (%)
4.1
6.9
Table 2. LLOQ, dynamic range and accuracy