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Application News No. C153 First Edition: Aug. 2 017 For Research Use Only. N ot for use i n diagnostic p rocedure. This publication may contain references to products that are not available in your country. Please contac…

Application

News

No.

C153



Validation Test of the Fully Automated

Pretreatment Analysis Method

Calibration curves were created from the control blood

plasma with standards added and the integrity of

accuracy and precision were evaluated based on the

analysis results of the QC samples (at concentrations of

n = 5) (Table 2). Good linearity was obtained in the set

concentration range for all antiarrhythmics. The

accuracy of the QC samples in the entire range,

including the quantitative lower limit, was within 100

± 15 %. Similarly, precision (%RSD) was within 15 % and

good repeatability was obtained.

Immediately after analysis of the highest calibration

standard sample, blank blood plasma was measured to

check for carryover in the fully automated sample

preparation LC/MS/MS system. No significant carryover

was detected for any of the drugs upon comparison

with the peak intensity of the lowest calibration

standard sample (Fig. 3).

The above results show that the fully automated

sample preparation LC/MS/MS system used in this

article is capable of sufficiently reliable quantitative

analysis when performing consecutive analyses of

samples of wide-ranging concentrations.

Table 2 Validation Test Results for Simultaneous Analysis of Antiarrhythmic Drugs and Metabolites

Compounds

Cal. Range

[ng/mL]

Correlation

Coefficient

Accuracy

Precision

%RSD, n=5

LLOQ Low Medium High LLOQ Low Medium High

Amiodarone

100-3000 0.9983 98.3 100.6 99.4 103.9 4.1 2.9 3.0 2.7

Desethylamiodarone

100-3000 0.9987 99.2 98.9 101.1 100.3 5.3 4.2 3.6 4.2

Bepridil

50-1500 0.9992 100.9 100.5 96.6 103.4 4.1 3.7 2.3 1.8

Flecainide

50-1500 0.9987 98.1 98.7 96.7 101.4 4.7 3.3 2.4 2.4

Pilsicainide

100-3000 0.9987 100.4 99.6 97.3 104.8 4.0 3.0 1.8 2.0

Cibenzoline

50-1500 0.9987 102.4 101.4 99.1 102.9 4.2 3.4 3.0 2.4

Mexiletine

100-3000 0.9984 104.5 107.4 106.3 107.8 3.8 3.9 2.6 2.6

*1: 100 ng/mL for LLOQ, 250 ng/mL for Low, 1000 ng/mL for Medium, 3000 ng/mL for High

*2: 50 ng/mL for LLOQ, 125 ng/mL for Low, 500 ng/mL for Medium, 1500 ng/mL for High

Carryover Test Results

Table 3 Analysis Conditions (Validation Test)

stem : CLAM-2000 + Nexera + LCMS-8060

Protocol : Plasma disp. 15 μL - acetonitrile disp. 285 μL - shaking at 1900 rpm, 120 sec - filtration for 90 sec

Column : Shimadzu GLC Mastro C18 (50 mmL. × 2.1 mmI.D., 3 μm)

Mobile Phase : A) 0.1% Formic acid - Water, B) 0.1% Formic acid - Methanol

Flow Rate : 0.4 mL/min

Time pro

ram : B Conc. 10% (0 min) – 100% (2 – 3.5 min) – 10% (3.51 – 6 min)

Column Temp. 50 °C In

ection Volume : 0.2 μL

Probe Volta

e : 2.0 kV (ESI-positive mode)

Interface Temp. : 300 °C DL Temp. : 250 °C

Block Heater Temp. : 400 °C Nebulizing Gas Flow : 3 L/min

Heatin

Gas Flow : 10 L/min Dr

Gas Flow : 10 L/min

Carry Over Test:

Analysis of Blank Plasma Following

The Highest Calibration Standard Sample

Application

News

No.

C153



Comparative Test with Manual Pretreatment

A comparative test was performed between a manual

pretreatment method and the fully automated

pretreatment analysis method that employs the fully

automated sample preparation LC/MS/MS system.

Human blood plasma for measuring the concentration

of amiodarone was used.

The manual pretreatment method involved manually

isolating the blood plasma, adding acetonitrile, and

mixing to perform deproteinization. After centrifugal

separation of this sample, the supernatant was then

transferred to a vial for LC/MS/MS analysis. On the

other hand, the fully automated pretreatment analysis

method enabled the entire analysis process, from

blood plasma isolation to LC/MS/MS analysis, to be

performed completely automatically using the system

described in this article (Fig. 4).

A comparison of quantitative values between the

methods was performed for amiodarone and the

metabolite desethylamiodarone (Fig. 5 and 6, Table 4

and 5). In the wide range of concentrations detected

from the samples, there was favorable agreement

between the quantitative results of the manual

pretreatment method and the fully automated

pretreatment analysis method. The coefficient of

determination (R2) of both methods calculated from

these results was 0.95 or higher (Fig. 7 and 8).

The fully automated pretreatment analysis method

used by this system is a fast and low-burden analysis

technique that achieves quantitative results equivalent

to conventional manual pretreatment methods and we

anticipate its utilization into the future.

Pretreatment Workflow of the Manual Pretreatment Method and Fully Automated Pretreatment Analysis Method

Human Blood Plasma (Sample 3) Analysis Results Using

the Manual Pretreatment Method

Human Blood Plasma (Sample 3) Analysis Results Using

the Fully Automated Pretreatment Analysis Method

1.50 1.75 2.00 2.25 min

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

(x100,000)

2:DEA 618.00>72.10(+) CE: -32.0

1:AMD 646.00>58.10(+) CE: -52.0

Manual

Pretreatment

Amiodarone

546 ng/mL

Desethylamiodarone

416 ng/mL

1.50 1.75 2.00 2.25 min

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

(x100,000)

2:DEA 618.00>72.10(+) CE: -32.0

1:AMD 646.00>58.10(+) CE: -52.0

Automated Pretreatment

with CLAM-2000

Amiodarone

557 ng/mL

Desethylamiodarone

423 ng/mL

Application

News

No.

C153

First Edition: Aug. 2017

For Research Use Only. Not for use in diagnostic procedure.

This publication may contain references to products that are not available in your country. Please contact us to check the availability of these

products in your country.

The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.

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publication to refer to either the entities or their products/services. Shimadzu disclaims any proprietary interest in trademarks and trade names

other than its own.

The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its

accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the

use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject

to change without notice.

www.shimadzu.com/an/

Table 4 Quantitative Results of the

Manual Pretreatment Method and

Fully Automated Pretreatment Analysis Method

(Amiodarone)

Amiodarone

Manual

[ng/mL]

Automated

[ng/mL]

Ratio %*

Sample 1 373 411 110.2

Sample 2 399 404 101.3

Sample 3 546 557 102.0

Sample 4 205 211 102.9

Sample 5 963 895 92.9

Sample 6 1,318 1,213 92.0

Sample 7 1,271 1,229 96.7

Sample 8 1,233 1,282 104.0

Sample 9 2,259 2,208 97.7

Avera

e 100.0

RSD % 5.8

* Automated Pretreatment / Manual Pretreatment

Comparison of Quantitative Results for Amiodarone

Table 5 Quantitative Results of the

Manual Pretreatment Method and

Fully Automated Pretreatment Analysis Method

(Desethylamiodarone)

Desethylamiodarone

Manual

[ng/mL]

Automated

[ng/mL]

Ratio %*

Sample 1 304 271 89.1

Sample 2 412 366 88.8

Sample 3 416 423 101.7

Sample 4 271 240 88.6

Sample 5 717 654 91.2

Sample 6 151 150 99.3

Sample 7 431 408 94.7

Sample 8 664 628 94.6

Sample 9 940 1,080 114.9

Avera

e 95.9

RSD % 8.9

* Automated Pretreatment / Manual Pretreatment

Comparison of Quantitative Results

for Desethylamiodarone

Table 6 Analysis Conditions (Comparative Test of Pretreatment Methods)

stem : CLAM-2000 + Nexera + LCMS-8040

Protocol : Plasma disp. 50 μL - acetonitrile disp. 225 μL - mixing at 1900 rpm, 120 sec - filtration for 90 sec

Column : Shimadzu GLC Mastro C18 (50 mmL. × 2.1 mmI.D., 3 μm)

Mobile Phase : A) 0.1% Formic acid - Water, B) 0.1% Formic acid - Methanol

Flow Rate : 0.4 mL/min

Time pro

ram : B Conc. 10 % (0 min) – 100 % (2 – 3.5 min) – 10 % (3.51 – 6 min)

Column Temp. 50 °C In

ection Volume : 0.1 μL

Probe Volta

e : 4.5 kV (ESI-positive mode)

DL Temp. : 250 °C Block Heater Temp. : 400 °C

Neb. Gas Flow : 3 L/min Dr

ing Gas Flow : 15 L/min

[Acknowledgments]

We would like to thank pharmacist Yuko Shimamoto of the Pharmacy Division at the National Cerebral and Cardiovascular Center Hospital

(National Research and Development Agency) in Japan for her significant cooperation in the investigation provided in this article.

References • Guidance for Industry : Bioanalytical Method Validation (2001, US FDA)

• Guideline on Bioanalytical Method Validation in Pharmaceutical Development (2013, Japan MHLW)

Notes • The product described in this document has not been approved or certified as a medical device under the Pharmaceutical and

Medical Device Act of Japan. It cannot be used for the purpose of medical examination and treatment or related procedures.

• The samples described in this document were all sampled and measured at the National Cerebral and Cardiovascular Center

Hospital in Japan. Permission was obtained regarding the publication of measurement data.

y = 0.975x

R² = 0.9946

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

Automated Pretreatment

[ng/mL]

Manual Pretreatment

[ng/mL]

AMD

y = 1.007x

R² = 0.9541

250

500

750

1000

1250

0 250 500 750 1000 1250

Automated Pretreatment

[ng/mL]

Manual Pretreatment

[ng/mL]

DEA