10_Application_News_No_LC_26_ADI_049

LC - 26 -A DI- 049 Liquid C hromatography Mas s Spectrometry Analysis of Vitamin A in serum using CLAM- 2000 /LC-MS/MS LC/MS ■ Introduction Vitamin A, is a group of unsaturated nutritional organic compounds that include …

LC-26-ADI-049

Liquid Chromatography Mass Spectrometry

Analysis of Vitamin A in serum using

CLAM-2000/LC-MS/MS

LC/MS

■ Introduction

Vitamin A, is a group of unsaturated nutritional organic

compounds that include retinol, retinal, retinoic acid, and

several provitamin A carotenoids (most notably beta-

carotene).[1][2] Vitamin A has multiple functions: it is important

for growth and development, for the maintenance of the immune

system and good vision.[3][4] Vitamin A is needed by the retina

of the eye in the form of retinal, which combines with protein

opsin to form rhodopsin, the light-absorbing molecule[5]

necessary for both low-light (scotopic vision) and color vision.[6]

Vitamin A also functions in a very different role as retinoic acid

(an irreversibly oxidized form of retinol), which is an important

hormone-like growth factor for epithelial and other cells.[7]

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

concentration of Vitamin A in biological samples due to its high

level of sensitivity and specificity however, manual sample

preparation often involves several complicated steps which can

lead to erroneous results.

Also, lengthy and tedious sample preparation procedures along

with large number of samples, make LC-MS/MS a less

desirable method. Automated sample preparation module has

been shown to eliminate human error, as well as increase

laboratory efficiency in terms of throughput, enabling LC-MS/MS,

to find its place in the labs.

The ability to analyze Vitamin A by LC-MS/MS (LCMS-8040,

Shimadzu) using automated sample preparation (CLAM-2000,

“For research use only. Not for use in clinical diagnostics”

Shimadzu) is used to process large sample sets (Fig. 1). The

CLAM-2000 (Clinical Laboratory Automation Module), has the

ability to perform a variety of steps appropriate for automated

sample preparation, like precipitation, filtration, heating, shaking,

and pipetting. This system is seamlessly integrated with the LC-

MS/MS, requiring no human involvement after loading the

biological samples into the sample chamber. We validated the

data acquired by CLAMP-2000/LC-MS/MS, an automated

method using a home brew method for analysis.

Blood

serum

Place sample

collection tube in

CLAM-2000

Fig. 1 CLAM – 2000 and LC-MS/MS 8040 System

0.00 0.25 0.50 0.75 1.00 1.25 min

0.0

1.0

2.0

3.0

(x100,000)

1:Vi tami n A T IC(+) 2018 0511 _S_00 00031 7_Vit A(0000 )_01.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00031 8_Vit A(0000 )_02.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00031 9_Vit A(0000 )_03.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00032 0_Vit A(0000 )_04.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00031 7_Vit A(0000 )_01.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00031 8_Vit A(0000 )_02.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00031 9_Vit A(0000 )_03.l cd

1:Vi tami n A T IC(+) 2018 0511 _S_00 00032 0_Vit A(0000 )_04.l cd

Sample is

transferred to

LC-MS/MS

Automatically.

Fully automated sample preparation

Dispending

Samples

Dispending

Reagents

Shaking

Filtering

Heating

Sample

Transfer

Results without human

intervention

LC-26-ADI-049

Fig. 2 Analytical Flow with Parallel Processing

■ Method

Vitamin A was analyzed using home brew method. Serum

sample was loaded directly into the automated sample

preparation system CLAM-2000.

The CLAM-2000 was programmed to perform protein

precipitation using acetonitrile followed by precipitation and

sample collection. Sample preparation involved taking 15 uL of

sample, adding to it 90 uL of precipitant solution. Following

filtration, the filtrated sample is then transported using an arm

from the CLAM-2000 to autosampler of LC-MS/MS for analysis

without any human intervention. In Fig. 2 the analytical

workflow which works parallel to sample analysis and sample

preparation is represented.

The LC-MS/MS instrument was equipped with an atmospheric

pressure chemical ionization source (APCI).

The sample was analyzed on column at 40°C with isocratic

mode with flow rate of 300 µL/min.

■ Analytical Conditions

The auto optimization feature of Lab Solution software was

used to optimize MS/MS conditions using Vitamin A standard.

The autosampler was used for injecting the standard.

LC-MS/MS conditions are described in Tables 1 & 2.

Two MRM transitions were recorded and summed for signal

integration.

The CLAM-2000 was programmed to perform sample

extraction by protein precipitation followed by filtration and

sample collection.

System

Column

Column Temperature

Mobile Phases

Flow Rate

Total Run Time

Injection Volume

System

Ionization

Probe Voltage

Temperature

Gas Flow

: Nexera X2 (high pressure binary gradient)

: Information available on request

: 40°C

: 0.1% Formic Acid in Methanol

: 300 µL/min

: 1.5 min

: 50 µL

: LCMS-8040

: APCI

: 4.5 kV (positive ionization)

: Interface: 350°C

Desolvation Line: 280°C

Heater Block: 300°C

: Nebulizing Gas: 3 L/min

Drying Gas: 15 L/min

Table 1 Analytical Conditions

MRM

Transitions

Name MRM CE Dwell Time (msec)

Vitamin A 269.15 > 93.00 -12 80

269.15 > 81.00 -21 80

Dwell Time

CID Gas

Pressure

: 63 msec

: 230 kPa

Quadrupole

Resolution : Q1: Unit Q3: Unit

Table 2 MS/MS Acquisition Parameters

Reagent

Dispensing

10 uL

Sample

15 uL

Reagent

Dispensing

90 uL

Shaking

50 secs

Filtration

150 secs

Sample Preparation 5.5 mins

LC-MS/MS

Analysis 1.5 mins

Sample Preparation 5.5 mins

Sample

Injection

LC-MS/MS

Analysis 1.5 mins

Sample

Injection

Sample

Injection

LC-26-ADI-049

Table 6 Estrone QC Results

y = 1.0052x - 10.904

R² = 0.9316

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

CLAM

-2000 prp. Vitamin A Conc. (ng/mL)

Manual prep. Vitamin A Conc. (ng/mL)

Vitamin A sample preparation manually v/s CLAM-2000

Vit A Lower Range Upper Range Median Linear (Vit A)

0.00 0.25 0.50 0.75 1.00 1.25 min

0.0

1.0

2.0

3.0

(x100,000)

1:Vitamin A TIC(+) 20180511_S_00000317_Vit A(0000)_01.lcd

1:Vitamin A TIC(+) 20180511_S_00000318_Vit A(0000)_02.lcd

1:Vitamin A TIC(+) 20180511_S_00000319_Vit A(0000)_03.lcd

1:Vitamin A TIC(+) 20180511_S_00000320_Vit A(0000)_04.lcd

1:Vitamin A TIC(+) 20180511_S_00000317_Vit A(0000)_01.lcd

1:Vitamin A TIC(+) 20180511_S_00000318_Vit A(0000)_02.lcd

1:Vitamin A TIC(+) 20180511_S_00000319_Vit A(0000)_03.lcd

1:Vitamin A TIC(+) 20180511_S_00000320_Vit A(0000)_04.lcd

■ Calibration Curve

The Vitamin A standards, were serially diluted over a desired

clinical range of 125 - 1000 ng/mL and were placed on CLAM-

2000 for sample preparation, the way we carried out for samples.

The calibration curves showed good linearity (r

>0.996) over a

desired clinical range of 125 - 1000 ng/mL for Vitamin A (Fig. 3)

and calibration levels TIC’s are shown in (Fig. 4).

■ Data correlation with Manual method

Analytical data obtained using manual and automated sample

preparation methods for 43 human serum samples of Vitamin A

with known concentrations were studied and compared. In fig. 5,

orange line, grey line and yellow line represents the lower range,

upper range and median respectively. Percentage of Total error

(%TE) was used to define the upper and lower range of the

analyte. %TE refers to the amount of error that is allowable

without invalidating the interpretation of a test result.

As per Westgard guideline %TE of Vitamin A is ±17.1%. The

blue colored are the data points of Vitamin A obtained by CLAM-

2000.The r

(regression) obtained is 0.93 and r (correlation co-

efficient) is 0.96. Maximum data points of Vitamin A are within

the %TE.

Fig. 5 Data Correlation Plot between Automated Sample Preparation (CLAM – 2000)

and Manual Method (n=43 Human Serum Sample)

Fig. 3 Calibration Curve of Vitamin A (L1 –L4)

Fig. 4 TIC (Total Ion Chromatogram) of Calibrator