Bile acids - LC-MS/MS - Scherer et al.

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 Pump 2.2 Autosampler 2.3 Column 2.4 Mass spectrometer 3 Data analysis and quantification 3.1 Software 3.2 Calibration and quantification 4 Method Validation 4.1 Accuracy 4.2 Precision 4.3 LOD 4.4 Recovery 5 Sample data 5.1 Mass spectra 5.2 Chromatogram  6 Reference

Sample preparation

• 10µL of a methanolic IS-mixture were added to plasma
• 30µL of 1mol/L HCl were added to serum/plasma
• protein precipitation was performed with 1mL acetonitrile, followed by 1min vortex-mixing
• after 15min of centrifugation (14,000×g), the supernatant was evaporated to dryness under reduced pressure
• samples were re-dissolved in 100µL methanol/water (1/1, v/v, containing 10mmol/L ammonium acetate and 0.1% ammonium hydroxide)
• after centrifugation the supernatant was used for analysis

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Human plasma or serum	100µl	2.4µmol/L D4-CA	10µl
		2.6µmol/L D4-DCA
		4µmol/L D4-CDCA
		0.6µmol/L D4-LCA
		2.6µmol/L D4-UDCA
		4.6µmol/L D4-GCA
		14µmol/L D4-GCDCA

Instrumentation and method

Pump

• Type: binary and isocratic pump (Agilent 1200)
• Mode: gradient elution
• Solvent(s): Solvent A methanol/water (1/1, v/v) containing 0.1% ammoniumhydroxide (25%) and 10mmol/L ammonium acetate (pH 9); Solvent B methanol containing 0.1% ammoniumhydroxide (25%) and 10mmol/L ammonium acetate (pH 9)
• column flow was directed only from 1.0 to 5.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered

• Gradient:
  

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.5	100	0
0.5	0.5	100	0
4.5	0.5	50	50
4.6	0.5	0	100
5.5	0.5	0	100
5.6	0.5	100	0
6.5	0.5	100	0

Autosampler

• Type: CTC Pal
• Injection volume: 5µl
• Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH
  

Column

• Type: NUCLEODUR C18 Gravity Macherey-Nagel
• length: 50 x 2.1 mm i.d., 0.5µm pre-column filter
• Particle size: 1.8 µm
• Column temperature: 50°C

Mass spectrometer

• Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
• Ionization mode: ESI negative
• Ionization voltage: -4500V
• Source temperature: 450°C
• Collision gas: Nitrogen
• Collision gas pressure: medium
• MS/MS-conditions: multiple reaction monitoring

Analyte	Precursor [m/z]	Product [m/z]	Collision energy [eV]
CA	407.3	407.3	−30
CDCA	391.3	391.3	−30
DCA	391.3	391.3	−30
LCA	375.3	375.3	−30
UDCA	391.3	391.3	−30
HDCA	391.3	391.3	−30
GCA	464.3	74	−72
GCDCA	448.3	74	−70
GDCA	448.3	74	−70
GLCA	432.3	74	−64
GUDCA	448.3	74	−70
GHDCA	448.3	74	−70
TCA	514.3	80	−116
TCDCA	498.3	80	−116
TDCA	498.3	80	−116
TLCA	482.3	80	−108
TUDCA	498.3	80	−116
THDCA	498.3	80	−116

Data analysis and quantification

Software

• Analyst 1.4.2.

Calibration and quantification

• calibration type: matrix calibration - addition of naturally occurring species
• species used for calibration
  

Analyte	Human serum/plasma up to (μM)
CA	4.4
CDCA	8
DCA	5.2
LCA	0.4
UDCA	4.7
HDCA	4.7
GCA	9.4
GCDCA	28.2
GDCA	3.2
GLCA	0.2
GUDCA	0.32
GHDCA	0.32
TCA	2.8
TCDCA	9.2
TDCA	0.77
TLCA	0.2
TUDCA	0.32
THDCA	0.32

Method Validation

Accuracy

89 - 111 %

Precision

CV: 4 - 11 %

LOD

below 10 nmol/L

Recovery

above 80%

Sample data

Mass spectra

Chromatogram 

Reference

• Scherer M, Gnewuch C, Schmitz G, Liebisch G. Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Nov 15;877(30):3920-5.