Calibrator
Volume of stock standard (μL)
Volume of ethanol (μL)
Final concentration (μg/mL)
Final dilution
6
200
–
6.0
–
5
200
200
3.0
1:2
4
333
667
2.0
1:3
3
–
667
0.67
1:3
2
–
667
0.22
1:3
1
–
667
0.07
1:3
2.
Controls: Chromsystems coenzyme Q10 plasma control Bi-Level I+II (Chromsystems, GmbH, Gräfelfing, Germany): Control material is prepared per the manufacturer’s instructions; 2.0 mL of deionized water is added to the lyophilized material and allowed to stand at least 15 min prior to use. A negative control is prepared utilizing 3.0 % bovine albumin .
2.5 Equipment and Supplies
1.
Instrumentation: Applied Biosystems Inc. API 3200 with Shimadzu Prominence 20A HPLC system equipped with Analyst software version 1.6.1. Operating parameters are described in Table 2a, b. Parameters have been optimized specifically for the API 3200 with Shimadzu 20A Prominence HPLC System; some variations would be expected as tune settings often vary slightly between instruments.
Table 2
ESI-LC-MS/MS operating conditions
A. HPLC | |||||
Column temp | 40 °C | ||||
Flow rate | 0.80 mL/min | ||||
Gradient | Step | Time (min) | Module | Event | Parameter (%) |
1 | 0.10 | Pumps | Pump “B” conc | 0.0 | |
2 | 0.50 | Pumps | Pump “B” conc | 25 | |
3 | 1.00 | Pumps | Pump “B” conc | 60 | |
4 | 1.10 | Pumps | Pump “B” conc | 60 | |
5 | 2.00 | Pumps | Pump “B” conc | 80 | |
6 | 2.10 | Pumps | Total flow | 0.85 mL/min | |
7 | 2.10 | Pumps | Pump “B” conc | 80 | |
8 | 2.80 | Pumps | Pump “B” conc | 100 | |
9 | 3.50 | Pumps | Pump “B” conc | 100 | |
10 | 3.70 | Pumps | Pump “B” conc | 0.0 | |
11 | 5.00 | System controller | Stop | ||
B. MS/MS settings | |||||
Scan | MRM | ||||
Polarity | Positive | ||||
Curtain gas | 20.0 psi | ||||
GS1 | 30.0 psi | ||||
GS2 | 35.0 psi | ||||
NC | 3.00 | ||||
CXP | 7.00 | ||||
EP | 10.0 | ||||
TEMP | 400.0
 Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
Get Clinical Tree app for offline access
| ||||