Direct LC/MS Analysis of Basic Drugs in Plasma by On-line pretreatment with Weak CationExchange 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 biosamples 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 columnswitching 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

Degasser

Mix er
2 1 3 6 4 5

Analytical Column

|||LCMS-2010EV
LIQUID CHROMATOGR APH -M ASS SPECTROMETER

SHIMADZU

Pump

Pump Degasser
Autosampler

Tee MAYI WCX

C1 C2

Pump Degasser

MS spectrometer D Pump
Oven

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).

Injection volume 2 L 5 L 10 L 20 L 50 L

Extraction time 0.37 min 0.51 min 0.62 min 0.69 min 1.21 min

uV (x1,000,000) 2.00 1.75 1.50 1.25 1.00

Injection volume 2 L, 5 L,10 L, 20 L, 50 L

0.75
0.50 0.25 0.00 0.0 0.5 1.0 1.5 2.0 2.5

Area
min

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

Retention time (min) Mobile phase 0.02%Formic Acid/ACN=95/5 0.02%Acetic Acid/ACN=95/5 0.02%Acetic Acid 10mM Ammonium Acetate/ACN=95/5 10mM Ammonium Acetate Water/ACN=95/5 Water 10mM Ammonium Acetate/ACN=95/5 (Online dilution with water) (Dilution factor :10) Dibucaine 0.23 0.64 6.02 4.0 >10.00 >10.00 >10.00 >10.00 Verapamil 0.23 0.70 4.71 4.0 >10.00 >10.00 >10.00 >10.00 Lidocaine 0.14 0.30 0.64 0.9 >10.00 2.79 >10.00 >10.00 Desipramine 0.18 0.38 0.89 1.6 >10.00 6.51 >10.00 >10.00 Amitriptyline 0.24 0.50 2.41 2.8 >10.00 >10.00 >10.00 >10.00

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).

1: Desipramine 2: Dibucaine 3: Lidocaine 4: Verapamil 5: Amitriptyline

1 2 4

Figure 3: MS chromatogram of 5 basic drugs spiked in human plasma; each concentration: 20ng/mL, Injection volume: 20 L.

2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 0 50 100 150 200

Lidocaine n=1 n=2 n=3 711696 713181 703238 719508 719461

Verapamil 1757291 1742033 1711805 1721872 1718240

Amitriptyline 1087215 1107764 1112317 1095871 1092197

Lidocaine Verapamil Amitriptyline

n=4 n=5 S.D.

6713.043177 18872.6241 10595.7699

Average
%RSD

713416.8

1730248.2

1099072.8

0.940970717 1.09074664 0.96406443

250

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

MAYI-WCX 1
3 2 4 5

1: Desipramine 2: Dibucaine 3: Lidocaine 4: Verapamil 5: Amitriptyline

MAYI-ODS 1
3

2 5 4

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 didnt require a high amount of cations in the mobile phase. This means the Shim-pack MAYIWCX 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 highresolution analysis with a combination of smaller particle ODS columns and an analytical column.

Acknowledgment
We would like to express our thanks to Yoshiaki Sato, Naoki Asakawa, the codevelopers of the system and Shim-pack MAYI-WCX at Eisai Co., Ltd.