Direct LCMS Analysis

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Direct LC/MS Analysis of Basic Drugs in Plasma by On-line pretreatment with Weak Cation- Exchange Pretreatment Column Masatoshi Takahashi, William Hedgepeth, Shimadzu Scientific Instruments Inc, Columbia, MD, USA

description

LCMS

Transcript of Direct LCMS Analysis

Direct LC/MS Analysis of Basic

Drugs in Plasma by On-line

pretreatment with Weak Cation-

Exchange Pretreatment Column

Masatoshi Takahashi, William Hedgepeth, Shimadzu Scientific

Instruments Inc, Columbia, MD, USA

Introduction

The Shim-pack MAYI-column series has been developed to enable on-line pretreatment of bio-

samples for HPLC. The outer surfaces of the silica particles are coated with methyl cellulose

polymer, and the inner surface of pores are chemically bonded with stationary phase such as C18

(ODS type), C8, strong cation group (SCX) and so on. The Shim-pack MAYI columns show the

high ability for protein extraction from plasma samples due to the size exclusion function and the

hydrophilic surface of the particles. Bio-samples can be pretreated automatically by a column-

switching HPLC (Co-Sense for BA system, Shimadzu) with the MAYI pretreatment column.

The Shim-pack MAYI-SCX has strong ion exchange groups as the stationary phase to trap basic

compounds efficiently. However, the column requires a high amount of salts in the mobile phase

for eluting drugs retained by ion exchange interactions; the high concentration of salts in the

mobile phase may produce some problems with the use of mass spectrometry.

A new pretreatment column, Shim-pack MAYI-WCX (prototype), which has weak cation-exchange

groups as stationary phase, has been developed for trapping of basic compounds in

consideration of LCMS use. It is expected that the WCX stationary phase allows elution of basic

drugs under weak acidic mobile phase that is moderate for MS spec. The retention ability and

pretreatment effect of the Shim-pack MAYI-WCX was evaluated for desipramine, dibucaine,

lidocaine, verapamil and amitriptyline by using a column-switching system (Co-Sense for BA

system, Shimadzu).

Experimental

System Configuration

The HPLC was a Shimadzu Prominence system with a SIL-20A autosampler, LC-20AD pump

systems, a DGU-20A5 degasser, a CTO-20AC oven and a FCV-12AH six-port flow changeover valve

(see Figure 1). A single-Q mass spectrometer, LCMS-2010EV (Shimadzu), was used for detection.

Shim-pack MAYI-WCX (prototype, 10 × 4.6 mm, Shimadzu) was used as the pretreatment column.

Quantitative analysis

On-line pretreatment conditions

Column: Shim-pack MAYI-WCX (prototype, 4.6mmI.D., 10mmL)

Mobile phase (C1)*1: 5mM ammonium acetate/acetonitrile=96/4

Mobile phase (C2)*2: water/acetonitrile =1/4

*1) For trapping of test drugs, *2) For washing

Mobile phase (D)*3: Water

*3) For on-line dilution

Total flow rate: 3.0mL/min (C:0.3mL/min, D:2.7mL/min)

Dilution factor: 10 fold

Extraction time: 2min

Column temp: 40℃

Analytical conditions

Column: Shim-pack XR-ODS (3.0mmI.D., 50mmL)

Mobile phase (A): A :0.1% formic acid in water

Mobile phase (B): B :0.1% formic acid in acetonitrile

Gradient: 12%B (0min-2.00min) → 30%B (9.00min)

→80%B (9.02min-10min) → 12%B(10.02min) → stop(13min)

Flow rate: 0.7mL/min

Column temp: 40℃Detection: ESI(+)

Figure 1: System Configuration

Analytical

Column

Autosampler MAYI

WCXPump

MS spectrometer

C2

A

B

D

Pump

Pump

Degasser

Degasser

4 5

32

61

C1

Mix

er

Tee

Oven

Degasser

Pump

|||LCMS-2010EVLIQUID CHROMATOGR APH -M ASS SPECTROMETER

SHIMADZU

Results

1. The elution curve of plasma proteins from the Shim-pack MAYI-WCX was monitored

at UV 280nm to measure protein extraction efficiency. The time required for protein

extraction is listed in Figure 2. We regarded the time at which the elution curve fell to

a level of 5mAbs as the extraction time. The Shim-pack MAYI-WCX required 0.3 to

1.2 minutes for protein extraction when the injection volume was from 2 to 50 L.

2. The retention performance of basic drugs on the Shim-pack MAYI-WCX was

investigated for the purpose of controlling trap/elution of basic drugs (Table 1). The

basic drugs could be sufficiently trapped by use of neutral pH mobile phases. Acidic

mobile phases facilitated elution of the basic drugs from MAYI-WCX.

3. The test drugs were analyzed with good linearity and reproducibility by using

optimized conditions (Figures 3, 4).

4. The Shim-pack MAYI-WCX provided different selectivity from that of the Shim-pack

MAYI-ODS and showed good effect on removal of matrices in the plasma sample

(Figure 5).

5. When the Shim-pack MAYI-WCX was coupled to an analytical ODS column in the

column-switching system, the drugs were concentrated at the top of the analytical

ODS column at eluting conditions for the Shim-pack MAYI-WCX. As a result, the

combination provided very sharp peak shape (Figure 6).

Figure 2: Time for extraction of plasma proteins from Shim-pack MAYI-WCX,

dilution factor: 10 fold (Cflow/Dflow =1/7), loading solution: 10mM ammonium

acetate/acetonitrile=95/5, flow rate: 3.0mL/min

Injection volume Extraction time

2 L 0.37 min

5 L 0.51 min

10 L 0.62 min

20 L 0.69 min

50 L 1.21 min

0.0 0.5 1.0 1.5 2.0 2.5 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

uV (x1,000,000)

Injection volume

2 L, 5 L,10 L, 20 L, 50 L

Are

a

Table 1: Retention ability for the test basic drugs on MAYI-WCX column;

Retention time means elution time at peak initial point. Flow rate: 3.0mL/min

(total).

Mobile phaseRetention time (min)

Dibucaine Verapamil Lidocaine Desipramine Amitriptyline

0.02%Formic Acid/ACN=95/5 0.23 0.23 0.14 0.18 0.24

0.02%Acetic Acid/ACN=95/5 0.64 0.70 0.30 0.38 0.50

0.02%Acetic Acid 6.02 4.71 0.64 0.89 2.41

10mM Ammonium

Acetate/ACN=95/5

4.0 4.0 0.9 1.6 2.8

10mM Ammonium Acetate >10.00 >10.00 >10.00 >10.00 >10.00

Water/ACN=95/5 >10.00 >10.00 2.79 6.51 >10.00

Water >10.00 >10.00 >10.00 >10.00 >10.00

10mM Ammonium

Acetate/ACN=95/5 (Online dilution

with water)

(Dilution factor :10)

>10.00 >10.00 >10.00 >10.00 >10.00

Figure 3: MS chromatogram of 5 basic drugs spiked in human plasma; each

concentration: 20ng/mL, Injection volume: 20 L.

1: Desipramine

2: Dibucaine

3: Lidocaine

4: Verapamil

5: Amitriptyline

1

23

4

5

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

0 50 100 150 200 250

Lidocaine

Verapamil

Amitriptyline

Lidocaine Verapamil Amitriptyline

n=1 711696 1757291 1087215

n=2 713181 1742033 1107764

n=3 703238 1711805 1112317

n=4 719508 1721872 1095871

n=5 719461 1718240 1092197

S.D. 6713.043177 18872.6241 10595.7699

Average 713416.8 1730248.2 1099072.8

%RSD 0.940970717 1.09074664 0.96406443

Conc. (ng/mL)

Figure 4: Calibration curves (each 2, 10, 20, 100, 200ng/mL, 20 L injection)

and reproducibility (each 200ng/mL, 20 L injection) of 3 drugs in spiked

human plasma, lidocaine: r=0.9975, verapamil: r= 0.9991, amitriptyline:

r=0.9992

MAYI-WCX

MAYI-ODS

Figure 5: Comparison of MS TIC chromatogram of human plasma sample

pretreated by MAYI-WCX or MAYI-ODS

1: Desipramine

2: Dibucaine

3: Lidocaine

4: Verapamil

5: Amitriptyline

MAYI-WCX

MAYI-ODS

1

23

4

5

1

23

45

Figure 6: Comparison of MS chromatogram of 5 basic drugs in spiked human

plasma sample pretreated by MAYI-WCX or MAYI-ODS; each concentration:

20ng/mL, Injection volume: 20 L.

Conclusions

1. The Shim-pack MAYI-WCX column provided the good advantage of

matrices clean-up in bio-samples.

2. Basic drugs trapped on the Shim-pack MAYI-WCX could be eluted at

conditions of weak acid mobile phase, and the column didn’t require a high

amount of cations in the mobile phase. This means the Shim-pack MAYI-

WCX has high compatibility with MS spec.

3. The Shim-pack MAYI-WCX showed excellent ability for trapping amine

compounds in plasma samples.

4. The Shim-pack MAYI-WCX has a high potential ability to realize high-

resolution analysis with a combination of smaller particle ODS columns and

an analytical column.

AcknowledgmentWe would like to express our thanks to Yoshiaki Sato, Naoki Asakawa, the co-

developers of the system and Shim-pack MAYI-WCX at Eisai Co., Ltd.