Take the Hurt out of Analyzing Pain Management Drugs with LC/MS/MS and an Ultra II® Biphenyl Column

By Amanda Rigdon, Innovations Chemist

Analyze target compounds that can’t be retained on phenyl hexyl columns.
Fast, reliable analysis of a wide variety of pain medications.
Improved retention and good peak shape increase accuracy and reduces risk of matrix effects.

Chronic pain significantly reduces overall quality of life and is a common cause of disability. While mild symptoms can be alleviated with nonsteroidal anti-inflammatory drugs (NSAIDs), severe cases may be treated with prescribed opiate and opioid drugs, often in conjunction with NSAIDs and benzodiazepines. Analysis of these pain medications is often done using GC/MS, but in recent years labs are transferring methods to LC/MS/MS, since sample extraction and derivatization are not always necessary when using LC/MS/MS. Although LC methods have advantages, problems arise with compounds that are not well-retained on conventional reversed phase columns (e.g. C18). Phenyl phases can be a better alternative, but not all phenyl columns offer equivalent performance.

Separate a Wide Range of Drugs Quickly

Ultra II® Biphenyl columns provide greater retention than other phenyl columns and produce the fast, reliable separations needed for routine analysis. Early eluting compounds are well-retained and resolved from one another, while still maintaining a fast analysis time (Figure 1). In contrast, morphine, codeine, and their metabolites are not adequately retained and cannot be accurately quantified on a Phenomenex Gemini® C6-Phenyl column due to ion suppression arising from eluting in the column void volume with matrix interferences. Although the earlier-eluting compounds are retained much longer on the Ultra II®Biphenyl column, the overall elution time for all compounds, including more strongly retained benzodiazepines, is only about one minute longer. In order to achieve acceptable retention for the early-eluting compounds on the Gemini® C6-Phenyl column, a much longer run would be necessary.

Simplify the analysis of pain medications by using Ultra II® Biphenyl columns. These unique phenyl columns outperform competitors and easily retain and resolve target analytes from potential isobaric matrix interferences.

Figure 1: Target drug compounds can be quickly resolved on Ultra II® Biphenyl columns.

A: Ultra II® Biphenyl (Restek)

	RT (min.)	MRM2	MRM3
1. Acetaminophen	1.18	152/110	152/65
2. Morphine	1.79	286/157	286/181
3. Codeine	3.21	300/165	300/215
4. Oxycodone	3.46	316/298	316/241
5. Hydrocodone	3.60	300/199	300/171
6. Fentanyl	6.61	337/132	337/216
7. Buprenorphine	6.66	468/187	468/84
8. Lorazepam	6.79	321/275	321/229
9. Diazepam	8.06	285/154	285/193
10. Methadone	8.21	310/77	310/223

Column:

Ultra II® Biphenyl (cat.# 9609352)

Dimensions:

50mm x 2.1mm ID

Particle Size:

3µm

Pore Size:

100Å

Temp.:

40°C

Sample:

Conc.:

5 ng/mL in 1:10 urine:mobile phase

Inj. vol.:

5µL

Mobile phase:

A: 0.1% formic acid in water

B: 0.1% formic acid in methanol

Time (min.)	Flow (mL/min.)	%A	%B
0	0.5	90	10
10	0.5	0	100
10.1	0.5	90	10
12	0.5	90	10

Detector:

Det.:

Applied Biosystems/MDS Sciex LC/MS/MS

Model #:

API-5000™

Ion source:

TurboIonSpray®

Curtain gas:

25 psi (172.4 kPa)

Gas 1:

60 psi (413.7 kPa)

Gas 2:

40 psi (275.8 kPa)

Source temp.:

550°C

Source Voltage:

2000 V

Mode:

MRM

Dwell time:

50 ms

Instrument:

Applied Biosystems/MDS Sciex LC/MS/MS System

Acknowledgement:

Special thanks to Applied Biosystems for providing instrument time.

LC_CF0516

B: Phenyl hexyl (Phenomenex)

	RT (min.)	MRM2	MRM3
1. Morphine	0.28	286/157	286/181
2. Codeine	0.43	300/165	300/215
3. Oxycodone	0.50	316/298	316/241
4. Hydrocodone	0.55	300/199	300/171
5. Acetaminophen	0.91	152/110	152/65
6. Fentanyl	2.70	337/132	337/216
7. Buprenorphine	3.18	468/187	468/84
8. Methadone	3.73	310/77	310/223
9. Lorazepam	5.80	321/275	321/229
10. Diazepam	6.46	285/154	285/193