9_Application_News_No_SCA_210_051

Liquid Chromat ography Mass Spectr ometr y Analysis of thiaminpyrophosphat e and pyridox al - 5’ - phosphate in whole blood using fully automated sample preparation LC/MS/MS system (CLAM- 2000 + LCMS - 8045)  Introduct …

Liquid Chromatography Mass Spectrometry

Analysis of thiaminpyrophosphate and pyridoxal-5’-

phosphate in whole blood using fully automated sample

preparation LC/MS/MS system

(CLAM-2000 + LCMS-8045)

 Introduction

Vitamin B1, thiamin, plays an important role in the

metabolic pathway in the human body. The

biological active form is Thiamin Pyrophosphate

(TPP). The water soluble vitamin acts as a

coenzyme for the enzymatic degradation of

glucose in the citric acid cycle. A non-varied diet or

malnutrition can quickly lead to a deficiency which

can result in inter alia depression, muscle

weakness and tachycardia. Vitamin B6 has

multiple forms. The biological active form in the

human cell is Pyridoxal-5’-Phosphate (P5P). The

water soluble vitamin acts as a coenzyme in the

formation of amino acids, amines and peptides. In

case of a deficiency the other B vitamins will also

be deficient. A PLP deficiency can occur due to

chemotherapy, alcoholism, pregnancy and kidney

failure.

These two vitamins are predominately analysed

with HPLC and fluorescence detection, which

requires derivatization. More recent there is a lot of

development for the analyses of these vitamins

with LC-MS/MS and protein precipitation. Due to

rising number off samples an automated solution is

needed. The aim of these study was to set-up an

automated solution for the analysis of TPP and

P5P in whole blood, using the kit from

Instruchemie in combination with the CLAM-2000

and LCMS-8045.

 Materials and Methods

The quantitative analysis of TPP and P5P from

whole blood samples was performed using reagent

provided in Instruchemie Vitamine B1&B6 kit.

Chromatography separation was developed using

Mobile Phase 1 (ref 3156) and Mobile Phase 2 (ref

3157) and a Shimpack GIST C18-AQ column (ref

227-30765-03). Both vitamins and their internal

standards were monitored using a LC-MS/MS

system (Nexera X2 and LCMS-8045, Shimadzu,

Kyoto) following specific MRM transitions.

Sample preparation was performed using

precipitation reagent (ref 3169) and internal

standard mix (ref 3153). Analytical performance of

the method was monitored using whole blood

calibrators (ref 3168) and whole blood quality

control samples (ref 3159). Automatic sample

preparation was performed using CLAM-2000

(Shimadzu, Kyoto).

No. SCA_210_051

Figure 1: Fully automated Sample Preparation

LC-MS/MS system

 Fully automated sample preparation

With the aim to reduce the operator involvement,

to increase the throughput and data quality, the

manual sample preparation procedure was

substituted by an automatic procedure using a

novel Clinical Laboratory Automated sample

preparation Module (CLAM-2000) online with the

LC-MS/MS system (LCMS-8045).

Blood collection tubes were loaded together with

disposable micro-vial containing calibrators and

quality control samples. Precipitation reagent and

ISTD mix are placed into specific instrument slot

(8 ºC). The fully automated sample preparation

procedure contains all steps from sample

collection to LC-MS/MS analysis (Figure 2).

 Results

Figure 3 shows a representative mass

chromatogram of TPP and P5P and their internals

standards from a control sample at low level.

Calibration curves were prepared by continuous

analysis with fully automated sample preparation

and analysis and used to asses accuracy and

precision. Good linearity was obtained across the

set calibration range for both vitamins, with

accuracy of 100 ± 15% over the entire

measurement range including limit of

quantification.

SCA_210_051

Figure 2: Schematic preparation/analysis workflow

Figure 3: Low control sample TPP/P5P 49/31 nmol/L

TPP y=0.00420734x + 0.0902752

= 0.9973214

P5P y=0.00656861x + 0.0295692

= 0.9954683

Figure 4: Calibration curves for TPP (39 – 890

nmol/L) and P5P (21 – 550 nmol/L)

www.shimadzu.eu

Shimadzu Europa GmbH

Precision was measured at a % RSD of within

15% showing that good reproducibility was

achieved. These results indicate that automated

sample preparation and analysis with the

CLAM-2000-LCMS-8045 system is suitable for the

analysis of the biological active forms of vitamin

B1 and B6 in whole blood samples.

 Conclusion

Results indicate that the fully automated sample

preparation LC/MS/MS system in combination with

the Instruchemie vitamine B1&B6 kit can eliminate

the risk of error of variability introduced by manual

sample preparation which is often a problem for

whole blood analysis. Also the results indicate that

the quick and high precision analytical workflow

from the CLAM-2000 system in combination with

the LCMS-8045 is a perfect solution for the high

amount of whole blood samples that are analysed

for TDP and PLP in the clinical chemistry.

SCA_210_051