Retinoid determination - Gunderson et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 HPLC-MS-MS 5.1.1 HPLC (Agilent 1100) 5.1.2 MS 4000 Q-Trap (Applied Biosystems) 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Recovery 7 Internal Standard 8 Reference

Abstract

Gunderson et al. described a high-througput ultrasensitive analytical method for the determination all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans retinol (atROH) in human plasma. It is characterized by a simple retinoid isolation as well as low detection limits.

Analysed Matrices

human plasma

Analytes

• all-trans-retinoic acid (atRA)
• all-trans-4-oxo-retinoic acid (at4oxoRA)
• 13-cis-4-oxo-retinoic acid (13c4oxoRA)
• 13-cis-retinoic acid (13cRA)
• all-trans-retinol (atROH)

Sample Preparation

• thawing of plasma samples in the dark (-80°C -> 4°C, overnight)
• vortexing and centrifugation (5 min, 3200 g)
• 100 μl aliquots onto microtiterplate
• addition of 200 μl working IS MPE solution
• thermosealing (AB-0384/240, AB-gene)
• vortexing (10 min) and centrifugation (10 min, 3200 g, 6 °C)
• 100 μl aliquots of supernatant for HPLC-MS analysis

All handling of samples was performed under red light!

Analytical Method

HPLC-MS-MS

HPLC (Agilent 1100)

column: rapid-resolution high-throughput Agilent ZORBAX SB-C18 (4,6 mm * 50 mm, 1,8 μm)

T (injector): 9 °C; T (column): 60 °C; 1100 wellplate autosampler; 1100 quaternary pump; 1100 micro-vacuum degasser; 1100 column oven; 1100 diode-array detector

mobile phase: CH3CN/H2O/HCOOH (10/90/0.1) for 1 min

CH3CN/H2O/MeOH/HCOOH (45/45/10/0,1) lineary ramped at 2 min

CH3CN/MeOH/HCOOH (90/10/0,1) lineary 2-6 min

flow rate: 1,8 ml/min (0-6 min); 2,5 ml/min (6-6,6 min)

injection volume: 100 μl; total run time : 7 min

MS 4000 Q-Trap (Applied Biosystems)

APCI positive mode; Q1: high resolution; Q3: low resolution

scan modus: MRM

nebulizer gas: 60 psig

curtain gas: 10 psig

collision gas: 7 psig

nebulizer current: 5

T: 450 °C

optimized parameters: DP (38 V); EP (4-13 V); CE (15-22 V); CXP (9-12 V)

dwell times: 10-180 ms

AB software: Analyst (1.4.1)

m/z 315.4→297 (4-oxo-RAs, dwell time 180 ms); 301.2→205 (RAs, dwell time 100 ms); 305.0→209 (IS, dwell time 10 ms) and 269.2→93 (retinol, dwell time 10 ms)

Method Validation

Accuracy

87 - 108 %

Precision

inter-day (12 d) better than 12,5 % CV; intra-day » 7 %

LOD

mLOD: 1-4 pg; cLOD: 30-60 pg/ ml

LOQ

0,1 -0,2 ng/ ml

Recovery

~80-105%

Internal Standard

10,11,14,15-13C4-atRA

Reference

• T. E. Gunderson, N. E. Bastian, R. Blomhoff Rapid Commun. Mass Spectrom. 2007, 21, 1176-1186.

Institute of Basic Medical Sciences; University of Oslo; P.O. Box 1046 Blindern; N-0316 Oslo; Norway.

e-mail: teg@vitas.no