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Application New s No. C 12 3 Fig. 2 shows the mass chromatogram for a control sample consisting of seven antiepileptic drugs and drug metabolites added to human blood serum. Because LC/ MS/MS can detect target drugs sele…

Application

News

No.

C123

Liquid Chromatography Mass Spectrometry

High-Throughput Optimization of Therapeutic Drug

Monitoring Using Fully Automated Sample Preparation

LC/MS/MS System (CLAM-2000 + LCMS-8040)

LAAN-A-LM-E094

Therapeutic drug monitoring (TDM) is a series of

processes where the blood concentration of drugs in a

patient is measured to determine the optimal dose and

method of administration for an individual based on

pharmacokinetic and pharmacodynamic analysis. TDM

is used during drug treatment with drugs that pose

administration management difﬁculties, such as drugs

with a narrow therapeutic range or with an effective

range and toxic range that are close to each other. High

performance liquid chromatography (HPLC) has been

the main analytical method used with TDM, but recently

liquid chromatography-mass spectrometry (LC/MS/MS)

is being used to improve analytical accuracy and

precision based on its superior selectivity.

LC/MS/MS normally requires sample preparation steps

such as deproteinization and dilution to analyze a blood

serum or blood plasma sample. These steps introduce

the risk of error or variability occurring based on

operator skill. The volume of work performed by an

operator also increases in accordance with the number

of samples. Therefore, the sample preparation process

can become the bottleneck of an analytical workﬂow

when analyzing a large number of samples.

n High-Throughput Analytical Workﬂow for

Antiepileptic Drug Analysis

We introduce an example simultaneous analysis of

seven antiepileptic drugs and drug active metabolites in

blood serum using a fully automated sample

preparation LC/MS/MS system.

Preparation of blood serum samples for analysis

normally requires deproteinization by the addition of

organic solvent, and then centrifugal separation of solid

components followed by supernatant recovery. The fully

automated sample preparation LC/MS/MS system only

Fully Automated Sample Preparation LC/MS/MS System

This article introduces the results of TDM using a fully

automated sample preparation LC/MS/MS system

comprised of the CLAM-2000 fully automated LCMS

sample preparation unit and the LCMS-8040 high

performance liquid chromatograph-mass spectrometer.

This system resolves the above-mentioned problems

associated with TDM, and achieves TDM research results

on a fast and high-precision analytical workﬂow.

requires placing of the blood collection tube in the

system, as the system performs all these preparation

steps automatically, followed by LC/MS/MS analysis

(Fig. 1).

Preparation of the next sample can also be performed

in parallel with LC/MS/MS analysis, which can greatly

reduce the time required for each sample analysis. In

our example, a per-sample cycle time including analysis

of 9 minutes is achieved.

Fig. 1

Workﬂow for Simultaneous Analysis of Antiepileptic Drugs in Blood Serum Using Fully Automated Sample Preparation LC/MS/MS System

Preparation of the

blood collection tube

9 min 9 min

MS analysis

7 min

MS analysis

7 min

MS analysis

7 min

Deproteinization step

8 min

Deproteinization step

8 min

Deproteinization step

8 min

Deproteinization using the CLAM-2000

Sample injection

Sample injectionSample injection

Setting of blood collection

tube and reagents in system

Serum

Sample

injection

• Freeze-dried

blood serum

• 30 µL

Filtration

• PTFE membrane

• Pore size:

0.45 µm

• Pressure:

-50 to -60 kPa

• Time: 150 sec

Reagent

injection

• Methanol

• 270 µL

Stirring

• Rotation

speed

1800 rpm

• Time:

150 sec

Application

News

No.

C123

Fig. 2 shows the mass chromatogram for a control

sample consisting of seven antiepileptic drugs and drug

metabolites added to human blood serum. Because LC/

MS/MS can detect target drugs selectively based on the

Calibration curves were prepared by continuous analysis

with fully automated sample preparation and analysis,

and used to assess accuracy and precision (repeatability).

Good linearity was obtained across the set calibration

curve range for each antiepileptic drug (Fig. 3), with

mass and structure of those drugs, the results show no

apparent interference from other constituents in the

blood serum.

accuracy within 100 % ±15 % over the entire

measurement range including the minimum limit of

quantification. Similarly, precision was measured at a

%RSD of within 15 %, showing that good repeatability

was achieved (Table 1).

Fig. 2 Mass Chromatogram of Seven Antiepileptic Drugs and Drug Metabolites in a Control Serum Sample

Fig. 3 Calibration Curves of Seven Antiepileptic Drugs and Drug Metabolites

2.00 2.50 3.00 3.50 4.00 4.50 min

0.00

0.50

1.00

1.50

2.00

(× 100,000)

Diazepam 285.15 > 153.95 (+)

Tiagabine 376.25 > 111.05 (+)

Carbamazepine 237.20 > 194.05 (+)

Topiramate 338.10 > 78.00 (-)

Carbamazepine 10-11-epoxide 253.15 > 180.05 (+)

Felbamate 239.20 > 117.00 (+)

Levetiracetam 171.15 > 126.10 (+)

Diazepam

Tiagabine

Carbamazepine

Carbamazepine 10-11-epoxide

Topiramate

Felbamate

Levetiracetam

r= 0.999

= 0.999

r999.0=

=0.999

r999.0=

=0.999

Levetiracetam

Felbamate

Topiramate

Carbamazepine

-10,11-epoxide

Carbamazepine

Tiagabine

Diazepam

Concentration Concentration Concentration

Concentration

Concentration Concentration

Area AreaArea

Area

250000

500000

750000

1000000

1250000

1500000

1750000

2000000

2250000

500000

1000000

1500000

2000000

2500000

3000000

100000

200000

300000

400000

500000

600000

700000

100000

200000

300000

400000

500000

1000000

2000000

3000000

4000000

5000000

25000

50000

75000

100000

125000

150000

175000

250000

500000

750000

1000000

1250000

1500000

0 250 500 750

0 250 500 75

0 2500 5000 7500

0 250 500 750

Application

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Table 1 Results of Validation Test for Simultaneous Analysis of Antiepileptic Drugs

Table 2 Analytical Conditions for Antiepileptic Drugs

Compounds

Range

(ng/mL)

QC samples concentration

(ng/mL)

Accuracy (%) % RSD (n=6)

LLOQ Medium ULOQ LLOQ Medium ULOQ LLOQ Medium ULOQ

Levetiracetam 10 - 750

10 100 750 94.6 106.1 99.2 3.42 1.23 1.98

Felbamate 25 - 1000 25 250 1000 98.6 101.8 99.6 6.28 1.88 1.50

Topiramate 500 - 10000 500 2500 10000 102.3 97.1 100.6 6.71 3.58 2.96

Carbamazepine-10, 11-epoxide 5 - 1000 5 100 1000 92.9 107.8 99.3 7.48 3.32 1.41

Carbamazepine 10 - 1000 10 100 1000 90.6 110.3 99.1 3.79 3.42 1.19

Tiagabine 50 - 1000 50 250 1000 98.5 101.9 99.6 1.95 2.00 1.26

Diazepam 5 - 1000 5 250 1000 98.1 102.4 99.5 4.61 1.50 1.53

Column : Inertsil ODS-4 (50 mm L. × 2.1 mm I.D., 2 μm)

Mobile Phase : A 10 mmol/L Ammonium acetate - Water

: B Methanol

Flowrate : 0.4 mL/min

Time Program : B. Conc. 3 % (0 - 0.5 min) - 90 % (3.0 - 5.0 min) - 3 % (5.01 - 7.0 min)

Column Temperature : 40 °C

Injection Volume : 1 μL

Probe Voltage : 4.5 kV / - 3.5 kV (ESI-positive / negative mode)

DL Temperature : 150 °C

Block Heater Temperature : 400 °C

Nebulizing Gas Flow : 3 L/min

Drying Gas Flow : 10 L/min

MRM Transition : Levetiracetam (+) m/z 171.15 > 126.10, Felbamate (+) m/z 239.20 > 117.00,

Carbamazepine-10,11-epoxide (+) m/z 253.15 > 180.05,

Carbamazepine (+) m/z 237.20 > 194.05, Tiagabine (+) m/z 376.25 > 111.05,

Diazepam (+) m/z 285.15 > 153.95, Topiramate (-) m/z 338.10 > 78.00

System Validation for Antiarrhythmic Drugs Analysis

TMD is used with a wide variety of drugs, and the

physicochemical properties of these drugs differ

individually. Therefore, confirming whether a given

series of standard operations, which includes the

process steps, tools, instruments and equipment used in

an analytical workﬂow, are appropriate for the target

drug is important for ensuring the analytical results

obtained are valid. We introduce an example validation

of sample preparation and analysis operations using

antiarrhythmic drugs with very different physicochemical

properties, and in particular very different hydrophilic

properties.

We chose the highly hydrophilic drug sotalol (partition

coefﬁcient: log P=2.6342) and the highly hydrophobic

drug amiodarone (log P=6.9326) and its active

metabolite N-desethylamiodarone were chosen, and

performed simultaneous analysis using the fully

automated sample preparation LC/MS/MS system

(Fig. 4).

Fig. 4 Mass Chromatogram of Three Antiarrhythmic Drugs and Drug Metabolite in a Control Serum Sample

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

0.0

1.0

2.0

3.0

4.0

5.0

(× 100,000)

DEA 618.00 > 72.20 (+)

AMD 646.00 > 58.20 (+)

Sotalol 273.10 > 133.00 (+)

Sotalol

Amiodarone

N-Desethylamiodarone