Challenge in Trying to Reach Femtogram per Milliliter (fg/mL) Sensitivity in Plasma
for the Quantification of a Cyclic Peptide: Desmopressin
Louis-Philippe Morin1, France Landry1, Jean-Nicholas Mess1, Kelli Jonakin2, Mauro Aiello2, Xavier Misonne2, Gary Impey2, Johnny Cardenas2 and Fabio Garofolo1 *
1Algorithme Pharma Inc., Laval (Montréal), QC, Canada
2AB SCIEX, Concord, ON, Canada
2.33
300
100
0.28
0
0.56 0.64
0.5
1.11
1.0
1.72
1.52
1.5
Time, min
2.0
XIC of +MRM (2 pairs): 535.400/328.200 Da from Sample 6 (CR09-P1A) of 1.wiff (Turbo Spray), Smoothed
Max. 740.0 cps.
1.79
1.91
700
DETECTION
• AB SCIEX QTRAP®5500 and QTRAP®6500
• MRM mode ESI(+)
• The [M+2H]2+ (m/z 535.4 → 328.2) was monitored for
desmopressin
RESULTS: SENSITIVITY COMPARISON
500
400
2.32
300
100
2.55
2.14
0.28
0.57
0.5
0.77
1.44
1.0
1.5
Time, min
2.0
2.5
C) Extracted ULOQ (250.00 pg/mL)
XIC of +MRM (2 pairs): 535.400/328.200 Da from Sample 3 (CR09-P10A) of 1.wiff (Turbo Spray), Smoothed
Max. 6.1e4 cps.
1.90
6.0e4
In order to evaluate if a novel generation of triple quadrupole
mass spectrometer would allow to reach fg/mL level of
sensitivity for the quantification of demopressin, standards
were prepared in crashed plasma matrix which was found to
reduce adsorptive losses. Post-extraction spiked approach
was used to limit any extraction variability and therefore to
allow for an unbiased comparison between the
QTRAP®5500 and the QTRAP®6500.
5.0e4
For sake of comparison, the samples were first injected on a
QTRAP®5500 under generic LC-MS/MS parameters. The
lower limit of detection (LOD) was found to be 80 fg oncolumn, which had a signal-to-noise ratio (S/N) of 4.5 with a
coefficient of variation (%CV) of 13.1% (n=4). This was
found too variable for quantification and therefore, the lower
limit of quantification was set at 160 fg on-column (S/N of
8.4 and %CV of 2.5, n=4).
4.0e4
3.0e4
2.0e4
1.0e4
0.0
0.5
1.0
1.5
Time, min
2.0
2.5
Figure 3: Calibration Curve for Desmopressin
Figure 4: LOD and LLOQ on the QTRAP® 5500
1.40
Calibrant
1.20
Using this extraction procedure and fast reversed phase
chromatography, an LLOQ of 2.00 pg/mL was achieved
within 3 minutes run time on a QTRAP®5500 (Figure 2).
The assay was linear (weighted 1/x2) over a range of 2.00
to 250.00 pg/mL (Figure 3). Precision and accuracy meet
acceptance criteria (Table 1)
1.71
0
RESULTS: DESMOPRESSIN BIOASSAY
A method for the quantification of the cyclic peptide
Desmopressin was developed in our laboratory using as
low as 250 µL of plasma samples. This method utilises a
specific and selective extraction on Oasis µELution plate
which allows to concentrate the analyte up to 3.3 fold.
Mean
S.D.
N
% C.V.
% Nominal
2.43
600
METHODS: SENSITIVITY COMPARISON
CHROMATOGRAPHY
• Agilent Technologies Series 1100 pumps and autosampler
(QTRAP®5500) and Shimadzu LC20AD (QTRAP®6500)
• Zorbax 300SB-C18, 50x2.1mm, 3.5µm
• Gradient elution of 0.1% HCOOH and ACN
2.5
B) Extracted LLOQ (2.00 pg/mL)
200
SAMPLE
• Desmopressin was spiked post extraction in crashed
plasma matrix
2.55
2.14
200
High QC
187.50
179.56
184.50
186.38
181.11
170.19
175.70
179.57
5.93
6
3.3
95.8
1.00
0.80
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
Nominal Back Calculated Deviation
pg/mL
pg/mL
%
2.00
1.92
-4.2
4.00
4.21
5.1
5.00
5.29
5.8
12.50
11.92
-4.6
37.50
35.58
-5.1
62.50
69.19
10.7
100.00
102.31
2.3
175.00
164.98
-5.7
212.50
199.95
-5.9
250.00
253.87
1.5
80 fg on-column
S/N = 4.5
%CV 13.1
0.60
Time (min)
0.40
Regression type: Linear 1/x2
Coefficient Correlation: r = 0.9953
0.20
0.00
0.00
50.00
100.00
150.00
Concentration (pg/mL)
200.00
250.00
160 fg on-column
S/N = 8.4
%CV 2.5
Figure 5: LOD and LLOQ on the QTRAP® 6500
Intensity (cps)
400
LOQ QC
2.00
1.91
2.10
2.07
1.74
1.98
2.21
2.00
0.16
6
8.2
100.0
Concentration (pg/mL)
Low QC
Mid QC
6.00
50.00
6.86
53.80
5.34
49.53
5.47
50.93
5.78
53.03
6.29
52.51
--51.47
5.95
51.88
0.63
1.55
5
6
10.5
3.0
99.2
103.8
The exact same samples were analyzed on a QTRAP®6500
to evaluate the IonDrive™ technology. The lower limit of
detection (LOD) was determined as 20 fg on-column which
had a S/N of 4.6 with a coefficient of variation (%CV) of 11.8%
(n=4). The lower limit of quantification was set at 40 fg oncolumn (S/N of 8.1 and %CV of 6.0, n=4). Data are
summarized in Table 2. Therefore, in the case of
desmopressin, the QTRAP® 6500 was deemed 4 times more
sensitive than the previous generation of tandem mass
spectrometer.
20 fg on-column
S/N = 4.6
%CV 11.8
Time (min)
Intensity (cps)
500
Intensity (cps)
Figure 1: Structure of Desmopressin
2.43
600
Intensity (cps)
Desmopressin is a synthetic cyclic peptide that is closely
related to the human hormone arginine vasopressin. It is
an antidiuretic used for the treatment of diabetes insipidus
and nocturnal enuresis. It acts by increasing water
reabsorption in the renal collecting ducts of the kidneys
and therefore reduces urine production. LC-MS/MS
assays for the quantification of desmopressin generally
involve laborious extraction and chromatographic
procedures in order to achieve low pg/mL quantification
limits. Moreover, large plasma sample volumes (1mL) are
often required for the analysis, which is not always
compatible with pre-clinical studies in animals or raises
safety concerns for human studies. In this research, the
IonDrive™ technology is used to reach low pg/mL
sensitivity and to attempt fg/mL quantification.
700
Intensity (cps)
INTRODUCTION
DETECTION
• AB SCIEX QTRAP®5500
• MRM mode ESI(+)
• The [M+2H]2+ was monitored for desmopressin
(m/z 535.4 → 328.2) and for the ISTD vasopressin
(m/z 542.9 → 328.2).
Max. 840.0 cps.
800
Intensity, cps
• Results
– An LLOQ of 2 pg/mL was reached for the
quantification of desmopressin in human plasma on
the QTRAP®5500.
– The QTRAP®6500 was found 4 times more sensitive
for the detection of desmopressin.
CHROMATOGRAPHY
• Agilent Technologies Series 1100 pumps and autosampler
• XBridge BEH300 C18, 50x2.1mm, 3.5µm
• Gradient elution in 2.4 minutes run time
Table 1: Within Run Precision and Accuracy for
Desmopressin
1.79
Peak Area Ratio
• Method
– Desmopressin was extracted from plasma by SPE
– Sensitivity comparison was performed on a
QTRAP®5500 and QTRAP®6500.
XIC of +MRM (2 pairs): 535.400/328.200 Da from Sample 5 (CR09-BLA) of 1.wiff (Turbo Spray), Smoothed
Intensity (cps)
SAMPLE EXTRACTION
• Range of 2.00 – 250.00 pg/mL in plasma
• 250 µL of sample extracted by solid phase extraction using
Oasis µElution SPE.
• Samples were eluted in a final volume of 75µL and 60µL
were injected.
Intensity, cps
• Purpose
– Use of IonDrive™ technology to attempt
desmopressin lower limit of quantification (LLOQ) at
fg/mL level.
Figure 2: Representative Chromatograms of Extracted
Desmopressin Samples Injected on the QTRAP®5500
A) Extracted Blank
Intensity, cps
METHODS:DESMOPRESSIN BIOASSAY
Intensity (cps)
OVERVIEW
40 fg on-column
S/N = 8.4
%CV 6.0
Time (min)
Table 2: Sensitivity Comparison Between QTRAP®5500
and QTRAP®6500
Desmopressin load
on-column
fg
20
40
80
160
QTRAP® 5500
S/N
%CV
n=4
4.5
8.4
QTRAP® 6500
S/N
%CV
n=4
5.3
11.8
8.1
6.0
13.1
2.5
CONCLUSION
According to the above results, the use of the IonDrive™
technology combined with an in-house-developed specific
clean & selective extraction/enrichment can allow the
quantification of desmopressin in plasma at fg/mL level. An
LLOQ of 500 fg/mL could be obtained on the QTRAP®6500
with as low as 250µL of plasma sample.
ACKNOWLEDMENTS
Algorithme Pharma would like to thank AB Sciex for their
availability and support throughout the QTRAP®6500
performance evaluation.
Time (min)
* CORRESPONDING AUTHOR