൮agement Drugs for Forensic Toxicology - Waters · was achieved between isobaric ... Phenyl XP...

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1 WATERS SOLUTIONS Xevo ® TQD ACQUITY UPLC ® I-Class System CORTECS ® C 18 2.7 µm, 3.0 x 50 mm Column (p/n 186007370) XBridge ® BEH Phenyl XP , 2.5 µm, 3.0 x 50 mm Column (p/n 186006069) MassLynx ® Software KEY WORDS Forensic toxicology, pain management, opiates, benzodiazepines, amphetamines, stimulants APPLICATION BENEFITS Rapid analysis of 35 forensic toxicology drugs Enhanced retention of polar compounds Improved resolution vs. competitive biphenyl columns Low backpressures compatible with traditional HPLC systems INTRODUCTION In forensic toxicology, drug screening panels often include such commonly used substances such as opiates, benzodiazepines and stimulants. These panels are often analyzed by LC-MS using traditional C 18 column technologies. Key considerations include the ability to chromatographically resolve the various pairs of isobaric compounds included in these panels, while maintaining good peak shape for a variety of compounds. In addition, when using traditional HPLC systems, the ability to analyze samples as rapidly as possible without exceeding the pressure limitations of the system is very important. This application note highlights the capabilities of Waters’ new CORTECS C 18 2.7 μm Columns and XBridge BEH Phenyl XP 2.5 μm Columns for this type of application. In the case of the CORTECS C 18 Column, the high efficiency packing of solid core 2.7 μm particles yields excellent performance that equals or exceeds competitive columns at lower operating backpressures. If alternative selectivity is desired, the phenyl functionality of the BEH phenyl column enhances the retention of opiate compounds. This enhanced retention can potentially result in reducing ion suppression from urinary matrix components. Both columns achieve excellent baseline separation between isomers, and the entire panel of 35 compounds, including opioids, benzodiazepines, stimulants, and other drugs of abuse can be analyzed in under 4 minutes at backpressures compatible with any HPLC system. Advantages of CORTECS C 18 2.7 μm and XBridge BEH Phenyl XP 2.5 μm Columns for the Analysis of a Comprehensive Panel of Pain Management Drugs for Forensic Toxicology Jonathan Danaceau, Erin Chambers, and Kenneth Fountain Waters Corporation, Milford, MA, USA

Transcript of ൮agement Drugs for Forensic Toxicology - Waters · was achieved between isobaric ... Phenyl XP...

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WAT E R S SO LU T IO NS

Xevo® TQD

ACQUITY UPLC® I-Class System

CORTECS® C18 2.7 µm, 3.0 x 50 mm

Column (p/n 186007370)

XBridge® BEH Phenyl XP,

2.5 µm, 3.0 x 50 mm Column

(p/n 186006069)

MassLynx® Software

K E Y W O R D S

Forensic toxicology, pain management,

opiates, benzodiazepines, amphetamines,

stimulants

A P P L I C AT IO N B E N E F I T S■■ Rapid analysis of 35 forensic

toxicology drugs

■■ Enhanced retention of polar compounds

■■ Improved resolution vs. competitive

biphenyl columns

■■ Low backpressures compatible with

traditional HPLC systems

IN T RO DU C T IO N

In forensic toxicology, drug screening panels often include such commonly

used substances such as opiates, benzodiazepines and stimulants. These panels

are often analyzed by LC-MS using traditional C18 column technologies. Key

considerations include the ability to chromatographically resolve the various

pairs of isobaric compounds included in these panels, while maintaining good

peak shape for a variety of compounds. In addition, when using traditional HPLC

systems, the ability to analyze samples as rapidly as possible without exceeding

the pressure limitations of the system is very important. This application note

highlights the capabilities of Waters’ new CORTECS C18 2.7 μm Columns and

XBridge BEH Phenyl XP 2.5 μm Columns for this type of application. In the

case of the CORTECS C18 Column, the high efficiency packing of solid core

2.7 μm particles yields excellent performance that equals or exceeds competitive

columns at lower operating backpressures. If alternative selectivity is desired,

the phenyl functionality of the BEH phenyl column enhances the retention of

opiate compounds. This enhanced retention can potentially result in reducing ion

suppression from urinary matrix components. Both columns achieve excellent

baseline separation between isomers, and the entire panel of 35 compounds,

including opioids, benzodiazepines, stimulants, and other drugs of abuse can be

analyzed in under 4 minutes at backpressures compatible with any HPLC system.

Advantages of CORTECS C18 2.7 μm and XBridge BEH Phenyl XP 2.5 μm Columns for the Analysis of a Comprehensive Panel of Pain Management Drugs for Forensic ToxicologyJonathan Danaceau, Erin Chambers, and Kenneth FountainWaters Corporation, Milford, MA, USA

2Advantages of CORTECS C18 2.7 μm and XBridge BEH Phenyl XP 2.5 μm Columns

E X P E R IM E N TA L

Stock solutions were obtained from Cerilliant

Corporation, Round Rock, TX. Stock solutions

were prepared in methanol. Working solutions

were prepared in 5% acetonitrile containing

0.1% formic acid.

LC conditions

LC system: ACQUITY UPLC I-Class,

Fixed Loop (FL) with

Column Manager (CMA)

Columns: CORTECS C18

2.7 µm, 3.0 x 50 mm

(p/n 186007370)

XBridge BEH Phenyl XP

2.5 µm, 3.0 x 50 mm

(p/n 186006069)

Column temp.: 30 ˚C

Sample temp.: 10 ˚C

Injection volume: 10 µL

Mobile phase A: MilliQ water with

0.1% formic acid

Mobile phase B: Acetonitrile with

0.1% formic acid

The mobile phase gradient is listed in Table 1.

MS conditions

MS system: Xevo TQD

Ionization mode: ESI Positive

Capillary voltage: 0.5 V

Collision energy: Optimized for individual

components

Cone voltage: Optimized for individual

components

Data management: MassLynx v 4.1

scn 855 Software

R E SU LT S A N D D IS C U S S IO N

Waters CORTECS C18 2.7 μm Column, an XBridge BEH Phenyl XP 2.5 μm Column,

and a competitor’s biphenyl core shell column (2.6 μm) were used to analyze a

panel of 35 common pain management compounds (Figure 1), including opioids,

benzodiazepines, stimulants, benzoylecgonine (BZE), and phencyclidine (PCP).

All columns had the same dimensions (3.0 x 50 mm). The solvent gradient is

listed in Table 1. The entire gradient cycle was 5 minutes.

All compounds eluted within 4 minutes and showed good, symmetrical peak

shape. Average peak width and maximum backpressure for all columns are

shown in Table 2.

Time (min) Flow (mL/min) % MPA % MPB

0.0 0.6 95 5

4.0 0.6 40 60

4.1 0.6 95 5

5.0 0.6 95 5

ColumnParticle size

(μm)Backpressure Mean peaks width (s)

CORTECS C18 2.7 2206 2.52

XBridge BEH Phenyl 2.5 3274 2.94

Competitor biphenyl 2.6 2492 2.71

Table 1. LC Gradient.

Figure 1. Compound key.

Table 2. Performance summary.

1) Amphetamine 2) MDA 3) Methamphetamine 4) MDMA 5) Phentermine 6) MDEA 7) BZE 8) PCP 9) Nitrazepam

10) Oxazepam 11) Alprazolam 12) Lorazepam 13) Clonazepam 14) Flunitrazepam 15) Temazepam 16) Diazepam

17) Morphine 18) Oxymorphone 19) Hydromorphone 20) Dihydrocodeine 21) Codeine 22) Oxycodone 23) 6-AM 24) O-desmethyl Tramadol 25) Hydrocodone 26) Norfentanyl 27) Tramadol 28) Normeperedine 29) Meperedine 30) Norbuprenorphine 31) Fentanyl 32) Buprenorphine 33) EDDP 34) Propoxyphene 35) Methadone

amines

benzodiazepines

opioids

3Advantages of CORTECS C18 2.7 μm and XBridge BEH Phenyl XP 2.5 μm Columns

Figure 2a. Chromatographic separation of opioids.

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

100

Time0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

100

Time0.40 0.60 0.80 1.00 1.20 1.40 1.60

%

0

100

17) Morphine 19) Hydromorphone 21) Codeine 25) Hydrocodone

17

25

19 21

17

25

19 21

17

25

19 21

CORTECS C18 2.7 µm,3.0 x 50 mm

XBridge BEH Phenyl2.5 µm, 3.0 x 50 mm

Competitor biphenyl2.6 µm, 3.0 x 50 mm

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

Time1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

Time1.00 1.50 2.00 2.50 3.00 3.50

%

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100

CORTECS C18 2.7 µm, 3.0 x 50 mm

Competitor biphenyl2.6 µm, 3.0 x 50 mm

17) Morphine 18) Oxymorphone 19) Hydromorphone 20) Dihydrocodeine

21) Codeine 22) Oxycodone 23) 6-AM 24) O-desmethyl Tramadol 25) Hydrocodone 26) Norfentanyl 27) Tramadol

28) Normeperedine 29) Meperedine 30) Norbuprenorphine 31) Fentanyl 32) Buprenorphine 33) EDDP

34) Propoxyphene 35) Methadone

XBridge BEH Phenyl 2.5 µm, 3.0 x 50 mm

17

20 21

19 22

24

23

25 26

27

30

31 29 28

32

34 33 35

18

1718 20,21 19

22 23

24

25 26

27

30

31

29 28

32

34 33

35

1718 20 21 19

22 23

24

25 26

27

30

31 29 28

32

34

33

35

A

B

The columns operated at backpressures well within the limit of traditional HPLC systems and, predictably, backpressure increased with

decreasing particle size. Interestingly, the CORTECS C18 Column, despite its larger particle size, demonstrated the best resolution, as

measured by average peak width (see Table 2). The chromatography of all opioid compounds is shown in Figure 2a and the separation of

key isobaric opiates can be seen in Figure 2b. All opioid drugs elute within 3.5 minutes and demonstrate good peak shape. As Figure 2b

shows, the isobaric pairs of morphine and hydromorphone (peaks 17 and 19), and codeine and hydrocodone (peaks 21 and 25) are well

separated on all columns. This is an important feature as these compounds must be resolved from each other for accurate identification and

quantification. While the BEH phenyl and biphenyl column both show increased retention of these compounds, which is most likely a result

of their phenyl functionality, excellent resolution is easily achieved on the CORTECS C18 Column.

2b. Chromatographic separation of key isobaric opiates.

4Advantages of CORTECS C18 2.7 μm and XBridge BEH Phenyl XP 2.5 μm Columns

Figure 3 shows the chromatography of the amines, PCP, and BZE. While all peaks demonstrate good peak

shape, the CORTECS C18 Column and XBridge BEH Phenyl XP Column both show excellent separation of these

compounds. Of particular note are methamphetamine and phentermine (peaks 3 and 5) which demonstrate

baseline separation on these two columns, but co-elute on the biphenyl column. This is an important feature as

these compounds have identical molecular formulas and both have a major fragment ion at m/z 91. The ability

to separate these compounds eliminates the risk of cross talk between these two stimulants and can be crucial

to unambiguous identification. Figure 3 also demonstrates that MDEA and benzoylecgonine (peaks 6 and 7),

which coelute on the biphenyl column, are separated on both the C18 and BEH phenyl columns.

Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100 1) Amphetamine 2) MDA 3) Methamphetamine 4) MDMA 5) Phentermine 6) MDEA 7) BZE 8) PCP

Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

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Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

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1 2

3 4

5

7 6

8

1

2

3

5

4

7 6

8

1 2

3 4

5 7 6

8

CORTECS C18 2.7 µm,3.0 x 50 mm

Competitor biphenyl2.6 µm, 3.0 x 50 mm

XBridge BEH Phenyl2.5 µm, 3.0 x 50 mm

Figure 3. Chromatographic separation of amines, BZE & PCP.

The benzodiazepine chromatography is shown in Figure 4. Good peak shape can be achieved on all columns.

Once again, the CORTECS C18 Column, despite its larger particle size, demonstrates the highest resolution for

this group of compounds (average peak width of 2.89 s).

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, T he Science of What’s Possible, CORTECS, XBridge, MassLynx, Xevo, and ACQUITY UPLC are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2014 Waters Corporation. Produced in the U.S.A. September 2014 720005185EN AG-PDF

CO N C LU S IO N

This application note highlights the analysis of a comprehensive

panel of opiates, benzodiazepines, and other drugs of abuse. Using

either Waters’ 2.7 μm CORTECS C18 Column, or a 2.5 μm XBridge

BEH Phenyl XP Column, all compounds were analyzed within

4 minutes with excellent peak shape and narrow peak widths.

Maximum backpressures were were respectively 2206 and

3274 psi, enabling the use of these columns on traditional

HPLC systems. Perhaps most importantly, baseline separation

was achieved between isobaric compounds, allowing for

their unambiguous identification and quantification. Whether

laboratories prefer the performance and efficiency of the solid-

core/superficially porous CORTECS C18 Column, or the unique

selectivity of the XBridge BEH Phenyl XP Column, each can be used

to rapidly analyze this important group of compounds.

Figure 4. Chromatographic separation of benzodiazepines.

Time2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

100

Time2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

%

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100

Time2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

%

0

100

9) Nitrazepam 10) Oxazepam 11) Alprazolam 12) Lorazepam 13) Clonazepam 14) Flunitrazepam 15) Temazepam 16) Diazepam

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10 11 13 12

15 14

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11

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12

15

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CORTECS C18 2.7 µm,3.0 x 50 mm

XBridge BEH Phenyl2.5 µm, 3.0 x 50 mm

Competitor biphenyl2.6 µm, 3.0 x 50 mm