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2 High throughput analysis for novel oral anticoagulants using LC-MS/MS system Integrated with automated sample preparation Introduction LC-MS/MS has become an essential tool for monitoring the concentration of drugs in …

PO-CON1602E

High throughput analysis for novel oral

anticoagulants using LC-MS/MS system

Integrated with automated sample

preparation

MSACL 2016 US

Daisuke Kawakami

1,2

, Taku Tsukamoto

1,2

, Naohiro Ohara

Kaori Shinmyozu

, Yuko Shimamoto

, Hikaru Shibata

1,3

Brian Field

, Yoshihiro Hayakawa

, Takafumi Tanigaki

Shin Nakamura

, Takeshi Kuwahara

Shimadzu Corporation, Kyoto, Japan,

National Cerebral and Cardiovascular Center, Osaka, Japan,

Shimadzu Scientic Instruments, Inc., Maryland, U.S.A.

High throughput analysis for novel oral anticoagulants using

LC-MS/MS system Integrated with automated sample preparation

Introduction

LC-MS/MS has become an essential tool for monitoring

the concentration of drugs in biological samples due to its

high level of sensitivity and specicity; however, manual

sample preparation often involves several complicated

manual steps which can introduce error into the results.

Additionally, the time consuming nature of the sample

preparation and the large number of samples makes

LC-MS/MS a less desirable method. Automated sample

preparation has been shown to eliminate human error, as

well as increase laboratory efciency, making LC-MS/MS a

feasible method to incorporate into workows requiring

high throughput.

In this study, we investigated the ability to analyze for

Novel Oral Anticoagulants (NOACs) by LC-MS/MS

(LCMS-8040, Shimadzu) using automated sample

preparation (CLAM-2000, Shimadzu) to process large

sample sets. The CLAM-2000 has the ability to perform a

variety of steps appropriate for automated sample

preparation by LC-MS/MS including precipitation,

ltration, heating, shaking, and pipetting. This system is

seamlessly integrated with the LCMS system requiring no

human involvement after loading the biological samples

into the sample chamber. We validated the automated

method by comparing the data collected on the

automated system to the data collected using a manual

sample preparation protocol. An additional consideration

we evaluated was the number of human hours dedicated

towards preparing samples for this assay using both

methods and the ability to incorporate a STAT sample

into the queue with rapid turn-around time for results.

Method

Plasma or plasma spiked with NOACs was loaded directly

into the CLAM-2000 for sample processing. The

CLAM-2000 was programmed to perform protein

precipitation using acetonitrile followed by ltration and

sample collection. The sample was then transported using

an arm from the CLAM-2000 to the HPLC for LC-MS/MS

analysis and no human intervention was required. For the

manual sample preparation, acetonitrile precipitation

followed by centrifugation was performed. After the

sample preparation the automated and manually

prepared samples were analyzed using the same

LC-MS/MS method.

Instruments and LC-MS/MS analytical condition

Fig.1 CLAM-2000 and LCMS-8040 system

High throughput analysis for novel oral anticoagulants using

LC-MS/MS system Integrated with automated sample preparation

System

Column : Mastro C18 2.1*100mm, 3μm

Column Temp. : 50 ºC

Mobile Phase A : 0.1% Formic Acid - Water

Mobile Phase B : 0.1% Formic Acid - Methanol

Time Program : 5%B(0-4 min) – 100%B(4-5min) – 5%B(5min)

Flow Rate : 0.4mL/min

Injection Volume : 2μL

Ionization : ESI Positive

[LC] NexeraX2

[MS] LCMS-8040

Table 1 LC-MS/MS condition

Plasma spiked with four NOACs (Apixaban, Rivaroxaban,

Edoxaban, Dabigatran) were used for calibration. Six

calibration standards, QC samples and human plasma

samples were prepared. These were precipitated in the

CLAM-2000 using acetonitrile and vacuum ltration. In

contrast, manual sample preparation used a

centrifugation step following precipitation to remove the

protein content. The ltrated sample was injected into

LC-MS/MS.

Sample preparation

Fig.2 Automated analysis from sample preparation to LC-MS/MS

460.20

436.10

548.20

472.00

Precursor (m/z)

443.10

145.00

152.20

289.05

Product (m/z)Polarity

Apixaban

Rivaroxaban

Edoxaban

Dabigatran

Compounds

MRM MS analysis

10min

Sample

Preparation

6min

Sample

Preparation

6min

MRM MS analysis

10min

Sample

Preparation

6min

12min 12min

Sample

injection

Filtration

• Time 120sec

Shaking

• Time 150sec

Reagent

Dispensing

• Acetonitrile

Sample

Dispensing

• Human plasma

Sample

injection

Sample

injection