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LC-MS/MS method for the determination of bile acid from serum sample using Cleanert MAS-MAW

Application Introduction

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Introductions

Bile acids are a class of structurally similar compounds that play essential roles in cholesterol homeostasis, lipid absorption, and intestinal signaling. Liver and gastrointestinal diseases can affect bile acid synthesis and disposition. For hepatic and intestinal diseases, serum or plasma bile acid concentrations have therefore long been utilized as prognostic and diagnostic markers. In line with the need for taking bile acid concentrations as biomarkers, a highly sensitive and rapid analysis method is needed to detect and measure bile acids and their conjugate in biological specimen. Liquid chromatography-mass spectrometry (LC-MS/MS) is an ideal option for the analysis of bile acids with the advantage of highly sensitivity.

Cleanert® MAS-MAW was used to purify the serum sample which with a mix mechanism of reverse phase and anion exchange interaction. It has proven to be an efficient and simple way to extract bile acid from serum sample. The mix-mode material eliminated matrix effect of serum such as proteins and phospholipids and obtained sufficient recovery with acceptable precision.


Analyte

Abbreviations

M.W.

R

R1

R2

R3

Ursodeoxycholic   acid

UDC

392.6

-H

-OH

-H

-OH

Cholic   acid

CA

408.6

-OH

-H

-OH

-OH

Chenodeoxycholic   acid

CDC

392.6

-OH

-H

-H

-OH

Deoxycholic   acid

DC

392.6

-H

-H

-OH

-OH

Lithocholic   acid

LC

376.6

-H

-H

-H

-OH

Tauroursodeoxycholic   acid dihydrate

TUDC

499.7

-H

-OH

-H

-NH(CH2)2SO3H

Taurocholic   acid sodium salt

TCS

537.7

-H

-OH

-OH

-NH(CH2)2SO3Na

Taurochenodeoxycholic   acid

TCDC

499.7

-OH

-H

-H

-NH(CH2)2SO3H

Figure 1 chemical structure of 8 bile acid and their taurine conjugates

Experimental

Material

A total of 8 bile acids were included in this experiment: cholic acid (CA), ursodeoxycholic acid (UDC), tauroursodeoxycholic acid dehydrate (TUDC), taurocholic acid sodium salt (TCS), chenodeoxycholic acid (CDC), deoxycholic acid (DC), taurochenodeoxycholic acid (TCDC), lithocholic acid (LC) were from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was prepared by a Milli-Q Academic System (Millipore, Billerica, MA, USA). Serum sample was obtained from laboratory rats. Formic acid and triethylamine were from Sigma Chemical (St. Louis, MO, USA). Methanol and acetonitrile were HPLC grade which purchased from merck (Darmstadt, Germany). Cleanert® MAS-MAW (100 mg/1mL) was from Bonna-Agela Technologies (Wilmington, Delaware, USA). The SPE vacuum manifold was from Bonna-Agela Technologies which used for sample preparation. The Nitrogen Evaporator was from Bonna-Agela Technologies which used for elutes concentration. The PTFE filtrations with the pore size of 0.22μm were from Bonna-Agela Technologies.

Instrumentation

The LC-MS/MS system was consisted of a Shimadzu LC-20A HPLC system with a binary pump and an auto-injector coupled with a triple quadrupole tandem mass spectrometer API4000+ (AB SCIEX, MA, USA). Data acquisition and processing were performed using Analyst software (version 1.5.1) from AB SCIEX. Chromatographic separation was achieved on a Durashell C18-AM column (particle size 3 μm, pore size 100 Å, 2.1 mm×100 mm, Bonna-Agela Technologies) at a column temperature of 30 °C. The gradient system was used with a mobile phase A (0.04% ammonia solution with 10mM ammonium acetate) and mobile phase B (acetonitrile), at a total flow rate of 0.25mL/min. The gradient system was started at 35% mobile phase B for 1minute, increased to 45% mobile phase B in 2 minutes, increased to 60% mobile phase B in 2.5 minutes, and decreased to 35% mobile phase B in 0.1 minute followed by 4.4 minutes re-equilibration. The total run time for each sample analysis was 10 minutes. The injection volume was 5 μL. The target compounds eluted from the HPLC column was introduced directly to the API4000+ and detected by the electrospray ionization interface with negative ion mode. Quantitative analysis was performed under MRM mode by calculating the peak areas. The ion source temperature was set at 500 °C. Nitrogen was used as curtain gas, GS1 and GS2 and set at 30, 60, and 55, respectively. The ion spray voltage was set at -4500V. The dwell time for all compounds was 100ms. The MS/MS transitions and optimal MS parameters were listed in Table 1.

Table 1 MS parameters and retention time of bile acid

Abbreviations

tR/min

Q1/Da

Q3/Da

DP/V

CE/V

CA

1.23

407.3

407.3

-130

-10

UDC

1.24

391.3

391.3

-145

-11

TUDC

1.35

498.4

498.4

-157

-9

TCS

1.36

514.3

514.3

-150

-7

CDC

1.72

391.4

391.4

-139

-8

DC

2.01

391.5

391.5

-130

-10

TCDC

2.36

498.3

498.3

-130

-10

LC

4.08

375.4

375.4

-127

-10

 

Sample preparation

100 μL of serum sample diluted with 100 μL of 1% formic acid aqueous solution was loaded into Cleanert® MAS-MAW which was pre-conditioned by 2 mL of acetonitrile and 2 mL of 3% formic acid aqueous solution sequentially. The cartridge was washed with 2 mL of methanol/ water (50/50, v/v), then discarded the eluate. The target compounds were eluted with 2 mL of methanol and 2 mL of triethylamine/ water/ methanol (2/10/88, v/v/v) sequentially. The eluates were collected together and concentrated at 40 °C under a gentle stream of nitrogen to dryness. The residues were reconstituted with 100 μL of methanol/ water (7/3, v/v) and filtrated by PTFE filtration with the pore size of 0.22 μm, and then analysis by LC-MS/MS.

Results and discussion

Cleanert® MAS-MAW is a mixed-mode mechanism cartridge with reverse phase and anion exchange material. Reverse phase interaction exist between polystyrene/ divinylbenzene materials and bile acids with the functional groups of carboxylic acid and hydroxyl. Anion exchange interaction was supplied by amino group which extracted the bile acids with sulphonate functional group.

Formic acid was used to adjust pH of loading sample, which could maintain a part of bile acids as molecular state. The step was important to get admired recovery data of bile acids with the functional groups of carboxylic acid and hydroxyl.

Most of the protein and phospholipids were washed out by 50% methanol in water without lose of bile acids. The elute process was divided into two steps. Reverse phase interaction was disrupted by 100% methanol in the first step which obtained bile acids with the functional groups of carboxylic acid and hydroxyl. In the second step, mixture of triethylamine and high percentage of methanol eluted the bile acids with functional group of sulphonate. Triethylamine as organic base was used in ion exchange SPE mode while ammonium hydroxide incompetent to restraint or ionize the target compound and absorbent material. In this application, triethylamine was used as ionization suppression to control the state of absorbent material.

The average recoveries of bile acid from the sample pretreatment method with two concentration levels at 50ng/mL and 500ng/mL are listed in Table 3.

Table 2 LOD of bile acid standard

Analyte

LOD/ng.mL-1

UDC

0.47

TUDC

1.51

TCDC

0.33

CA

0.16

DC

0.44

LC

2.39

CDC

0.90

TCS

1.58

 

Table 3 recovery data and precision of spiked sample

Spiked sample

CA

UDC

TUDC

TCS

CDC

DC

TCDC

LC

%Recovery-50ng/mL

106.00

114.70

95.20

116.10

115.00

109.10

100.00

116.40

%RSD(n=5)

7.08

6.62

5.48

5.69

5.72

6.96

5.31

7.48

%Recovery-500ng/mL

117.60

130.80

82.70

111.40

106.30

90.90

99.10

108.60

%RSD(n=5)

3.85

3.73

4.17

4.95

5.20

5.34

3.96

5.88

 

Figure 2 chromatogram of bile acid standard solution with the concentration of 500ng/mL

Figure 3 chromatogram of bile acid in serum

 

Figure 4 chromatogram of serum spiked with 500ng/mL of bile acid

Conclusions

This application has shown that Cleanert® MAS-MAW can be successfully used to extract bile acid from serum. The mixed-mode SPE plate could be used for eliminating matrix effect of phospholipids and proteins prior to the analysis of bile acid in serum by LC-MS/MS. A sufficient recovery and great precision were obtained. The method can be applied for the assay of bile acid in patient's serum samples.

Order Information

Products

Specification

Cat.No

Cleanert MAS-MAW

96-well   plate,100 mg/well

MAW0302-W

Filtration   membrane(PTFE)

φ13;   0.22 μm

AS041320

NV-96G   for 96 Well Plates

Adapt   to 96-well plate

NV-96G

Cleanert®   M96 Positive Pressure Device

Adapt   to 96-well plate

SPE-M96

Durashell C18 AM

2.1 × 15 0mm, 3 μm, 100 Å

DC931502-AM


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