Poster Number 075
Optimal Sensitivity and Increased Throughput Using a
Dual TIS/APCI Ionization Source and TurboFlow ®
Chromatography with LC/MS/MS
Doina Caraiman1, Tom Biesenthal1, Nadia Pace1, Shamim Haider1, Takeo Sakuma1, Lars Ynddal 2
Applied Biosystems/MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada, L4K 4V8;
2
Novo Nordisk A/S, Maaloev, Denmark
1
ABSTRACT
A sensitive, selective and fast method was developed for the analysis of Testosterone and Warfarin in
rat plasma from in vivo pharmacokinetic experiments. The instrumentation consisted of TurboFlow®
Chromatography (TFC) coupled with an API 4000TM mass spectrometer equipped with a combined TIS
(TurboIonSprayTM)/APCI source. Total assay time is as short as 4.3 minutes including direct online
injection of diluted plasma, HPLC separation and software-controlled switching of the ionization mode
during the chromatographic run. For each compound the optimal ionization method was determined
and set for the analytical run using the software. Limits of quantitation for Warfarin and Testosterone in
rat plasma are comparable with LOQs obtained for neat standards using conventional ion sources and
HPLC.
INTRODUCTION
There is an ever increasing burden on analytical laboratories to make high sensitivity measurements,
and to make them as quickly as possible. For the LC/MS analysis of compounds of widely differing
polarities the demands are even higher since compounds in the same sample can often only be ionized
optimally with the use of entirely different ionization sources (most commonly either electrospray
ionization or atmospheric pressure chemical ionization (APCI)). This study illustrates the use of a
combined TIS/APCI ionization source to combat this type of problem: as a software-selectable source,
the ionization mode can be changed on the fly, allowing the user to choose the best ionization mode for
each resolved compound in one analytical run. TurboFlow® Chromatography was used as a highthroughput method to eliminate the time-consuming sample preparation procedures for biological
samples.
MATERIALS AND METHODS
Instrumentation
- API 4000TM equipped with a DuoSprayTM ion source (Figure 1)
- Cohesive® 2300 pump/valve interface module (Cohesive® Technologies, Franklin, MA, USA) and
CTC PAL HTS autosampler (Cohesive® Technologies, Franklin, MA, USA) (Figure 2)
Sample Preparation
- Calibration standards were prepared by mixing 200 µL of rat plasma with 200 µL of standard
solutions in 10% methanol;
- Aliquots of 100 µL plasma samples from separate pharmacokinetic studies on rats for Testosterone
and Warfarin were mixed and diluted with 200 µL 10% methanol;
- Calibrators and unknowns were centrifuged at 16,600 g for 15 minutes. The supernatant was
transferred to 300 µL vials and loaded in the autosampler;
Chromatografic Conditions
- Extraction Column: CycloneTM HTLC (1.0 x 50 mm, 50 µm)
- Analytical column: Waters, C8 Nova Pak (4.6 x 20 mm, 4 µm)
- Mobile phase for loading pump and eluting pump
A: Water (0.1% HCOOH) : Methanol (95:5 v/v)
B: Water (0.1% HCOOH) : Methanol (5:95 v/v)
- Run time: 4.33 minutes
- Details for loading, transfer and eluting steps of the LC method: Figures 3-6
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ASMS 2003 – Poster Number 075: Caraiman, Biesenthal, Pace, Haider, Sakuma, Ynddal
Poster Number 075
Figure 1. DuoSprayTM source for API-4000TM
triple quadrupole mass spectrometer.
Figure 3. LC Methodology and Plumbing
Diagram: Loading Step
TurboIonSprayTM
(TIS) Inlet
APCI inlet
TurboIonSprayTM
Heater
Corona Discharge
Needle
TurboIonSprayTM
Probe
Figure 4. LC Methodology and Plumbing
Diagram: Transfer Step
The combined software-selectable TIS/APCI ionization
system offers the ability to quickly switch between the
TIS and APCI ionization techniques during a single
chromatographic run. The best ionization technique can
be chosen for each individual compound in a mixture of
drugs with significantly different polarities.1 Among the
benefits offered are: increased sensitivity over a larger
compound range, improved sample throughput and
accelerated method development.
Figure 5. LC Methodology and Plumbing
Diagram: Eluting Step
Table 1. MS method
Period
Compound
Duration
Warfarin
2.45
Ionization
Technique
TIS
Polarity
1
negative
MRM
transition
307.1 Æ 161.0
2
Testosterone
1.55
APCI
positive
289.1 Æ 97.2
Figure 2. Cohesive® 2300 System
Figure 6. LC Methodology and Plumbing
Diagram: Valve switching and Equilibration
The Cohesive® system combines on-line sample
preparation using TFC with analytical LC and has been
successfully used for dextromethophan metabolite studies
with direct injection of diluted rat plasma.2
Sample preparation for TFC is fast and much less labor
intensive compared to the traditional off-line sample
preparations such as liquid/liquid or solid phase extraction.
The dual column configuration was used to improve
sensitivity and selectivity of the assay.
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ASMS 2003 – Poster Number 075: Caraiman, Biesenthal, Pace, Haider, Sakuma, Ynddal
Poster Number 075
RESULTS
Figure 7. Warfarin and Testosterone
in rat plasma - typical chromatogram
Figure 8. Warfarin and Testosterone
in rat plasma - quantitation
Switch ionization mode
and polarity
Inject next sample
Warfarin
Testosterone
TIS negative
APCI positive
Warfarin
Testosterone
LOQ (fg on column)
68
630
Figure 9. Warfarin in rat plasma
Calibration Curve
Table 2. Warfarin in Plasma - Calibration
Expected
Mean calculated Standard Precision Accuracy
concentration concentration Deviation C.V. (%)
(%)
ng/mL
ng/mL (n=5)
0.05
0.049
0.004
9.12
97.26
0.10
0.106
0.002
2.09
106.44
0.50
5.00
10.00
0.480
5.014
10.001
0.009
0.067
0.043
1.90
1.33
0.43
96.02
100.27
100.01
Figure 10. Testosterone in rat plasma
Calibration Curve
Table 3. Testosterone in plasma - Calibration
Expected
Mean calculated Standard Precision Accuracy
concentration concentration Deviation C.V. (%)
(%)
ng/mL
ng/mL (n=5)
1
0.908
0.074
8.13
90.82
2
1.904
0.095
4.99
95.22
10
9.768
0.218
2.23
97.68
100
108.241
1.588
1.47
108.24
200
219.841
3.847
1.75
109.92
1000
990.004
16.427
1.66
99.00
2000
1982.333
9.445
0.48
99.12
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ASMS 2003 – Poster Number 075: Caraiman, Biesenthal, Pace, Haider, Sakuma, Ynddal
Poster Number 075
Figure 11. Pharmacokinetic study:
Warfarin i.v. 0.1 mg/kg and p.o. 0.2 mg/kg
Figure 12. Pharmacokinetic study:
Testosterone i.v. 5 mg/kg and p.o. 10 mg/kg
10000
1000
1000
Concentration (ng/mL)
Concentration (ng/mL)
Warfarin i.v. dose 0.1 mg/kg
Warfarin p.o. dose 0.2 mg/kg
Testosterone i.v. dose 5 mg/kg
100
10
Testosterone p.o. dose 10 mg/kg
1
100
0.1
0
50
100
150
200
250
300
350
400
Sampling Time, post dose (minutes)
0
50
100
150
200
250
300
350
400
Sampling Time, post dose (minutes)
CONCLUSIONS
Fast TIS/APCI switching allows TIS and APCI ionization for Warfarin and Testosterone, respectively
in one single acquisition run. Switching the ionization mode and the polarity are performed so that the
two peaks situated at less than 0.3 min apart in a chromatographic run can be baseline separated in
two different periods.
Cohesive® 2300 TurboFlow® Chromatography system was used to reduce the sample preparation
time for rat plasma samples from in vivo pharmacokinetic studies. Dual column focusing mode was
used for optimum sensitivity.
A high-throughput method combining the advantages of using the dual ion source in combination with
HTLC was developed for analysis of Warfarin and Testosterone from in vivo pharmacokinetic studies.
Quantitation limits comparable with results for neat standards analyzed with conventional HPLC
and ion sources, good precision (C.V. < 10%) and accuracy were obtained.
REFERENCES
1.Haider, S., Alary, J.-F, Kovarik, P. and Covey, T., poster at the Montreux Conference, Montreux, Nov. 2002.
2.Ynddal, L. and Hansen, S. H., accepted to J. Chromatography, 2003.
ACKNOWLEDGEMENTS
Many thanks to Cohesive Technologies for the loan of the Cohesive 2300 system and to Francois Espourteille and Sarah
Vannozzi from Cohesive Technologies for useful discussions.
TRADEMARKS/LICENSING
TurboIonSpray, and API 4000 are trademarks owned by Applera Corporation or its subsidiaries in the United States and
certain other countries and DuoSpray™ is a trademark owned by Applied Biosystems/MDS Sciex instruments. Applied
Biosystems and AB are trademarks of the Applera Corporation. MDS Sciex is a trademark of MDS Inc. Cohesive Technologies
is a registered trademark of Cohesive Technologies Inc. HTLC is a trademark of Cohesive Technologies Inc.
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ASMS 2003 – Poster Number 075: Caraiman, Biesenthal, Pace, Haider, Sakuma, Ynddal