ADV06-15_1-412

43 / 413

| 1-800-356-1688 or 1-814-353-1300 | Feedback? E-mail 17
A Fresh,
New Style
Coming Soon!
Figure 1: Analysis of 29 drug compounds and metabolites at 100 ng/mL in urine on an Ultra Biphenyl column.
response of the individual compound. All calibration curves employed
1/x weighting. As shown in Table I, good linearity was achieved with cor-
relation coefficient values exceeding 0.999 for most compounds.
LOQs were determined by evaluating signal-to-noise ratios for the
three transitions used for each compound, and values ranged from
1 ng/mL to 5 ng/mL for most compounds. Several analytes had LOQs of
10 ng/mL; only norbuprenorphine had an LOQ of 25 ng/mL, which was
expected since it is a poor responder and usually requires further sam-
ple preparation. With the exception of methadone, the quantification
ion for each compound had a signal-to-noise ratio of ≥10 at the LOQ,
and each qualifier ion had a signal-to-noise ratio of ≥3. Because metha-
done was a very high responder, the first two transitions for this drug
overloaded the detector at higher concentrations, so only the third tran-
sition was used for quantification. The first two transitions may be used,
but detuning these transitions is recommended to reduce response and
improve linearity.
Accuracy and Reproducibility
Accuracy and precision at the LOQ were assessed for each compound;
acceptable ranges were considered to be 90-110% recovery and ≤15%
coefficient of variation (CV). Accuracy ranged from 88% to 113% for all
analytes except norbuprenorphine, which typically is not determined
using a dilute-and-shoot method. Precision results ranged from 1% to
23%, and all compounds except for codeine, norbuprenorphine, and
sertraline had passing results of ≤15% CV for precision (Table I).
Since retention time shifts can be a source of downtime and sample
reanalysis, retention time reproducibility across multiple column lots
was also evaluated. Replicate injections of a 1 µg/mL solvent standard
were analyzed on three different lots of Ultra Biphenyl columns under
the same conditions used for the samples. Retention times for each
compound were determined and the maximum retention time varia-
tion across all three lots of analytical columns was just 0.13 minutes. This
indicates retention times are stable and predictable, which minimizes
the need to reset retention time windows when columns are changed.
Conclusion
Partial validation results indicate this method is suitable for the quantifi-
cation of a broad range of therapeutic drugs and metabolites in urine at
levels ranging from 1-1,000 ng/mL. By using a highly reproducible 5 µm
Ultra Biphenyl column and the multi-drug method conditions estab-
lished here, labs can reduce downtime and improve productivity.
For additional clinical/forensic articles, visit
LC_CF0535
Peaks RT (min)
1. Morphine 0.95
2. Oxymorphone 1.08
3. Pregabalin 1.29
4. Hydromorphone 1.34
5. Gabapentin 1.56
6. Codeine 2.16
7. Codeine-d3 (IS) 2.16
8. Oxycodone 2.29
9. Hydrocodone 2.33
10. 7-Aminoclonazepam 2.49
11. Tapentadol 2.52
12. Zopiclone 2.52
13. Norbuprenorphine 2.62
14. 7-Aminoflunitrazepam 2.65
15. Zolpidem 2.69
16. Citalopram 2.87
17. Fentanyl 2.87
18. Buprenorphine 2.89
19. Doxepin 2.92
20. Doxepin-d3 (IS) 2.92
21. Paroxetine 2.95
22. Promethazine 2.97
23. Nortriptyline 3.02
24. Amitriptyline 3.07
25. EDDP 3.08
26. Lorazepam 3.08
27. Sertraline 3.09
28. Methadone 3.11
29. Clonazepam 3.17
30. Flunitrazepam 3.31
31. Diazepam 3.37
32. Diazepam-d5 (IS) 3.37
Column:Ultra Biphenyl (cat.# 9109512); Dimensions: 100 mm x 2.1 mm ID; Particle Size: 5 µm; Pore Size: 100 Å; Temp.: 30 °C; Diluent:
Water:acetonitrile (90:10) + 0.1% formic acid; Conc.: 100 ng/mL (final dilution = 20x); Inj. Vol.: 30 µL;Mobile Phase:A: Water + 0.1%
formic acid, B: Acetonitrile + 0.1% formic acid; Gradient (%B): 0 min (10%), 1.00 min (10%), 3.5 min (100%), 4.0 min (100%), 4.1 min (10%),
5.5 min (10%); Flow: 0.6 mL/min; Detector: AB SCIEX API 4000 MS/MS; Model #: API 4000; Ion Source: TurboIonSpray®; Ion Mode: ESI+;
Ion Spray Voltage: 3000 kV; Curtain Gas: 40 psi (275.8 kPa); Gas 1: 60 psi (413.7 kPa); Gas 2: 60 psi (413.7 kPa); Interface Temp.: 600 °C;
Mode: MRM; Instrument:API LC-MS/MS. Notes: A 5 µm, 10 mm x 2.1 mm Ultra Biphenyl guard column (cat.# 910950212) was used in
conjunction with this analysis.
Sample Preparation:
- Fortify urine at 100 ng/mL.
- To 1 mL of urine, add 1 mL of 100 mM ammonium acetate (pH = 5.6) containing 2,000 units of β-glucuronidase from E. coli
(Sigma-Aldrich cat# G7396).
- Incubate for 90 minutes at 37 °C.
- Centrifuge at 3,000 rpm for 15 minutes.
- Dilute 100 µL of sample with 900 µL of water:acetonitrile (90:10) + 0.1% formic acid containing 4 ng/mL internal standard.
(Total dilution factor = 20x)
Ultra Biphenyl Columns (USP L11)
Physical Characteristics:
particle size: 3 µm or 5 µm, spherical
pore size: 100 Å
carbon load: 15%
endcap: fully endcapped
pH range: 2.5 to 8
temperature limit: 80 °C
Description cat.#
5 µm Columns
100 mm, 2.1 mm ID 9109512
For MRM transitions, visit and enter LC_CF0535 in the search.