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Rapid Analysis of 17 Bile Acids in Human Plasma
by LC-MS/MS

Abstract

Quantitative analysis of bile acids in plasma is critical for diagnosing liver disease as well as assessing drug safety. Accurate reporting can be difficult because of analyte characteristics, matrix effects, and other factors. Here, we establish a rapid, robust, and selective LC-MS/MS method for the analysis of bile acids in human plasma using a Restek Raptor C18 column. One of the significant improvements over other reported methods was the baseline separation of all 17 bile acids in 6 minutes, including three isomer groups.

Introduction

Bile acids are a group of major catabolic products of cholesterol, and they are critical signaling molecules that regulate cholesterol and glucose. The analysis of bile acids in human plasma is an important diagnostic tool as bile acids are biomarkers of liver disease and are also used as indicators of potentially harmful side effects of new drugs. There are two main types of bile acids based upon their functional groups: free (or unconjugated) bile acids and conjugated bile acids, primarily glycine- or taurine-bound (Figure 1, Table I). Quantitation of bile acids in matrix can be very challenging due to a number of factors. These include structural similarities, varying polarity and stereochemistry, the presence of isomers, limited fragmentation of unconjugated bile acids in a mass spectrometer, high endogenous levels, and matrix effects caused by phospholipids or triglycerides.

Traditionally, the analysis of bile acids in human plasma takes from 20 to over 60 min and results in only minimum resolution of isomers. In this study, a simple UHPLC method was developed for the simultaneous quantification of the major bile acids, as well as their glycine and taurine conjugates in human plasma. Using a Raptor C18 column and the methodology established here, baseline resolution and good chromatographic and quantitative results were obtained for all compounds in a fast, 6-min separation with a total analysis time of only 8.5 min.

Figure 1: Base Chemical Structure


Table I: R Group Identifications for Figure 1

Compound Name

R

R1

R2

R3

R4

R5

Ursodeoxycholic acid (UDCA)

H

H

H

OH

OH

OH

Hyodeoxycholic acid (HDCA)

OH

H

H

H

OH

OH

Cholic acid (CA)

H

OH

H

OH

OH

OH

Chenodeoxycholic acid (CDCA)

H

H

H

OH

OH

OH

Deoxycholic acid (DCA)

H

OH

H

H

OH

OH

Dehydrolithocholic acid (DHLCA)

H

H

H

H

OH

=O

Lithocholic acid (LCA)

H

H

H

H

OH

OH

Glycoursodeoxycholic acid (GUDCA)

H

H

H

OH

NHCH2CO2H

OH

Glycocholic acid (GCA)

H

OH

H

OH

NHCH2CO2H

OH

Glycochenodeoxycholic acid (GCDCA)

H

H

H

OH

NHCH2CO2H

OH

Glycodeoxycholic acid (GDCA)

H

OH

H

H

NHCH2CO2H

OH

Glycolithocholic acid (GLCA)

H

H

H

H

NHCH2CO2H

OH

Tauroursodeoxycholic acid (TUDCA)

H

H

H

OH

NHCH2CH2SO3H

OH

Taurocholic acid (TCA)

H

OH

H

OH

NHCH2CH2SO3H

OH

Taurochenodeoxycholic acid (TCDCA)

H

H

H

H

NHCH2CH2SO3H

OH

Taurodeoxycholic acid (TDCA)

H

OH

H

H

NHCH2CH2SO3H

OH

Taurolithocholic acid (TLCA)

H

H

H

H

NHCH2CH2SO3H

OH


Experimental

Calibration Standards and Quality Control Samples

Multicomponent calibration standards and fortified QC samples were prepared at the concentrations shown in Table II. All analytes and internal standards were added to plasma prior to the protein precipitation extraction step described below.

Table II: Calibration Standard and Quality Control Sample Concentrations (ng/mL)

Std 8

Std 7

Std 6

Std 5

Std 4

Std 3

Std 2

Std 1

High

Medium

Low

LLOQ

LCA

200

180

120

70

20

10

4

2

160

100

40

2

CA

100

90

60

35

10

5

2

1

80

50

20

1

CDCA

400

360

240

140

40

20

8

4

320

200

80

4

DCA

100

90

60

35

10

5

2

1

80

50

20

1

UDCA

400

360

240

140

40

20

8

4

320

200

80

4

HDCA

400

360

240

140

40

20

8

4

320

200

80

4

DHLCA

100

90

60

35

10

5

2

1

80

50

20

1

TLCA

1200

1080

720

420

120

60

24

12

960

600

240

12

TCA

1200

1080

720

420

120

60

24

12

960

600

240

12

TCDCA

1200

1080

720

420

120

60

24

12

960

600

240

12

TDCA

1200

1080

720

420

120

60

24

12

960

600

240

12

TUDCA

1200

1080

720

420

120

60

24

12

960

600

240

12

GLCA

600

540

360

210

60

30

12

6

480

300

120

6

GCA

600

540

360

210

60

30

12

6

480

300

120

6

GCDCA

600

540

360

210

60

30

12

6

480

300

120

6

GDCA

600

540

360

210

60

30

12

6

480

300

120

6

GUDCA

600

540

360

210

60

30

12

6

480

300

120

6


Sample Preparation

A 50 μL aliquot of 2x charcoal stripped human plasma (K2EDTA) and 50 μL of internal standards were protein precipitated using 800 μL of ice-cold acetonitrile. After vortexing and centrifugation at 4200 rpm for 10 min, the supernatant was transferred to a new vial and dried down at 60 °C under nitrogen. All samples were reconstituted in 200 μL of 35% methanol in water.

Instrument Conditions

LC-MS/MS analysis of bile acids in human plasma was performed on a Shimadzu Nexera UHPLC paired with a Shimadzu LCMS-8060 MS/MS. Instrument conditions were as follows, and analyte transitions are provided in Table III.

Analytical column:

Raptor C18 1.8 µm, 50 mm x 2.1 mm (cat.#. 9304252)

Guard column:

UltraShield UHPLC precolumn filter (cat.# 25810)

Mobile phase A:

5 mM ammonium acetate in water, pH unadjusted

Mobile phase B:

Methanol:acetonitrile (v/v, 50:50)

Gradient:

Time (min)Flow Rate (mL/min)%B

 

0.000.535

 

2.000.540

 

2.500.545

 

3.500.550

 

4.600.555

 

5.700.580

 

5.900.8*85

 

6.500.8*85

 

6.510.535

 

8.500.535

*Flow rate:

The flow rate was increased to 0.8 mL/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.

Injection volume:

3 µL

Column temp.:

60 °C

Ion mode:

Negative ESI


Table III: Ion Transitions and Internal Standards

Compound

MRM Transitions

Internal Standard

Ursodeoxycholic acid (UDCA)

391.4-391.4

UDCA-d4

Hyodeoxycholic acid (HDCA)

391.4-391.4

UDCA-d4

Cholic acid (CA)

407.3-407.2

CA-d4

Chenodeoxycholic acid (CDCA)

391.4-391.4

CDCA-d4

Deoxycholic acid (DCA)

391.4-391.4

DCA-d4

Dehydrolithocholic acid (DHLCA)

373.3-373.3

LCA-d5

Lithocholic acid (LCA)

375.5-375.3

LCA-d5

Glycoursodeoxycholic acid (GUDCA)

448.4-74.1

GDCA-d4

Glycocholic acid (GCA)

464.3-74.2

GCA-d4

Glycochenodeoxycholic acid (GCDCA)

448.4-74.1

GDCA-d4

Glycodeoxycholic acid (GDCA)

448.4-74.1

GDCA-d4

Glycolithocholic acid (GLCA)

432.3-74.0

GLCA-d4

Tauroursodeoxycholic acid (TUDCA)

498.4-80.1

TUDCA-d4

Taurocholic acid (TCA)

514.4-80.0

TCA-d5

Taurochenodeoxycholic acid (TCDCA)

498.4-80.1

TCDCA-d5

Taurodeoxycholic acid (TDCA)

498.4-80.1

TCDCA-d5

Taurolithocholic acid (TLCA)

482.4-80.0

TLCA-d4


Results and Discussion

Chromatographic Performance

An efficient baseline separation of 17 bile acids in human plasma was achieved in 6 minutes, including three groups of isomers (Figure 2). Note that the structure of the nucleus and side chains of bile acids affects their hydrophobicity, which governs their retention on a reversed-phase C18 column. As a result, the general elution order is tri-hydroxy bile acids (e.g., CA) first, followed by di-hydroxy bile acids (e.g., DCA and CDCA) and mono-hydroxy bile acids (e.g., LCA). Because the position and stereochemistry of hydroxyl groups can also influence the retention time, there are cases where di-hydroxy bile acids (e.g., UDCA) elute earlier than tri-hydroxy ones (e.g., CA). In addition, column pressure was monitored over the course of the experiments and showed no significant increase after at least 100 injections.

Figure 2: Chromatograms of 17 Bile Acids and Three Isomer Groups for the Analysis of Bile Acids in Human Plasma

Bile Acids with Isomer Separation in Human Plasma on Raptor C18 (1.8 μm) by LC-MS/MS
LC_CF0712
ColumnRaptor C18 (cat.# 9304252)
Dimensions:50 mm x 2.1 mm ID
Particle Size:1.8 µm
Pore Size:90 Å
Guard Column:UltraShield UHPLC precolumn filter 0.2 µm (cat.# 25810)
Temp.:60 °C
Sample
Diluent:70:30 Water:methanol
Inj. Vol.:3 µL
Mobile Phase
A:5 mM Ammonium acetate in water
B:50:50 Acetonitrile:methanol
Time (min)Flow (mL/min)%A%B
0.000.56535
2.000.56040
2.500.55545
3.500.55050
4.600.54555
5.700.52080
5.900.8*595
6.500.8*595
6.510.56535
8.500.56535
DetectorMS/MS
Ion Mode:ESI-
Mode:MRM
InstrumentUHPLC
Notes*The flow rate was increased to 0.8 mL/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.

Linearity

Method linearity was verified by analysis of an 8-point calibration curve (n=3). 1/x weighted linear regression was used for all compounds. Representative standard calibration curves are shown in Figure 3. The method has a dynamic range of 100-fold and excellent correlation coefficients (0.9989-0.9999) as shown in Table IV.

Figure 3: Representative Standard Curves.


Accuracy and Precision

Accuracy and precision were assessed at four different concentrations (LLOQ, QC-low, QC-medium, QC-high). Six replicates of each QC point were analyzed for intraday accuracy and precision. Blank matrix samples were extracted and analyzed to confirm that the endogenous levels of bile acids in the charcoal stripped plasma were below the LLOQ. Method accuracy, defined as the percentage of the measured concentration relative to the known concentration, ranged from 91.4% to 110% for the LLOQ, and from 92.6% to 107% across the low, medium, and high QC levels. The precision of the method, presented as relative standard deviation (%RSD), ranged from 3.02% to 16.3% for the LLOQ, and from 0.654% to 7.63% for all other QC levels combined. These results demonstrate that the method is accurate and precise over the range studied for the quantitative analysis of bile acids in human plasma (Table V).

Matrix Effect

Matrix effect caused by phospholipids was observed during method development. It was greatly reduced by extending the gradient and increasing the flow rate to thoroughly flush the column after the elution of all analytes.

Table IV: Linearity of Bile Acids in Human Plasma (1/x Weighted Calibration Curves)

Compound

Linear Range (ng/mL)

Regression Equation

R

GUDCA

6-600

y=0.9519x+0.00347

0.9996

TUDCA

6-600

y=43.072x+0.20729

0.9999

GCA

6-600

y=11.9x+0.047

0.9996

TCA

12-1200

y=15.17x+0.1077

0.9998

UDCA

4-400

y=1.0767x+0.00239

0.9997

HDCA

4-400

y=39.9x+0.217

0.9999

CA

1-100

y=11.9x+0.047

0.9989

GCDCA

6-600

y=0.9351x+0.0035

0.9997

TCDCA

12-1200

y=22.63x+0.0132

0.9997

GDCA

6-600

y=0.9875x+0.00435

0.9997

TDCA

12-1200

y=22.93x+0.000065

0.9998

CDCA

4-400

y=25.60x +0.7625

0.9995

DCA

1-100

y=21.09x+0.2392

0.9996

GLCA

6-600

y=1.0639x+0.00757

0.9998

TLCA

12-1200

y=0.5153x+0.00748

0.9997

DHLCA

1-100

y=1.349x+0.00248

0.9998

LCA

2-200

y=1.267x+0.01181

0.9999


Table V: Accuracy and Precision of QC Samples

Elution Order

Compound

QC-LLOQ (n=6)

QC-Low (n=6)

QC-Medium (n=6)

QC-High (n=6)

Precision (%RSD)

Accuracy (%)

Precision (%RSD)

Accuracy (%)

Precision (%RSD)

Accuracy (%)

Precision (%RSD)

Accuracy (%)

1

GUDCA

8.25

94.3

2.68

101

1.08

101

2.21

100

2

TUDCA

4.14

102

1.02

101

1.21

97.0

1.21

103

3

GCA

11.0

102

3.06

100

1.82

94.2

1.81

102

4

TCA

7.83

92.0

7.42

99.5

6.00

96.9

7.63

99.1

5

UDCA

7.78

95.7

5.82

104

4.23

102

6.04

107

6

HDCA

11.8

93.0

4.60

103

3.32

102

5.22

105

7

CA

16.3

91.4

4.58

102

3.21

98.3

3.23

104

8

GCDCA

7.83

103

1.79

103

2.30

97.9

1.05

102

9

TCDCA

6.25

110

3.12

102

3.12

94.9

7.08

98.3

10

GDCA

7.62

96.4

1.66

102

2.46

97.2

1.87

102

11

TDCA

4.89

106

3.43

99.0

3.39

92.6

7.06

97.7

12

CDCA

3.02

95.8

1.81

104

3.86

98.0

4.28

104

13

DCA

11.7

94.6

4.37

104

5.36

99.9

6.85

94.6

14

GLCA

6.93

101

0.833

101

1.14

97.9

1.66

102

15

TLCA

7.48

91.6

1.66

99.6

1.01

97.0

1.75

101

16

DHLCA

5.24

101

0.654

99.2

1.92

96.9

2.80

104

17

LCA

6.06

101

1.41

99.4

1.98

97.2

0.738

103


Conclusion

A rapid, robust, and selective LC-MS/MS method was established and verified for the analysis of bile acids in human plasma using a Restek Raptor C18 column. One of the significant improvements over other reported methods was the baseline separation of all 17 bile acids in 6 minutes including three isomer groups. Excellent linear correlation (R = 0.9989-0.9999), accuracy (91.4%-110%) and precision (0.654%-16.3%) results were obtained for all compounds across all QC levels. Matrix effects caused by phospholipids were greatly reduced by column flushing.

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