A Generic LC-MS Method for the Analysis of Multiple of Drug of Abuse Classes with the Thermo Scientific Exactive TM System

(1)

A Generic LC-MS Method for the Analysis of

Multiple of Drug of Abuse Classes with the Thermo

Scientific Exactive

TM

_System

Kent Johnson

(2)

List of drug of abuse candidates for LC-MS analysis

Benzodiazepines

Opiates

Other drugs group 1

Other drugs group 2

7-Aminonitrazepam

7-Aminoclonazepam

7-Aminoflunitrazepam

2-Hydroxy-ethyl-flurazepam

Desalkylflurazepam

Diazepam

Hydroxy-alprazolam

Hydroxy-triazolam

Nordiazepam

Lorazepam

Oxazepam

Temazepam

Morphine

Hydromorphone

Oxymorphone

Codeine

Dihydrocodeine

Hydrocodone

Oxycodone

Meperidine

Normeperidine

Ketamine

Norketamine

Butorphanol

Fentanyl

Norfentanyl

Nalbuphine

Alfentanil

Sulfentanil

Zolpidem

Trazodone

Venlafaxine

Zopiclone

Methylphenidate

Ritalinic Acid

Dextromethorphan

Dextrophan

Propoxyphene

Norpropoxyphene

6-MAM

(3)

Methods Employed Prior to LC-MS

• _{Benzodiazepines GC-MS}

• _{Opiates GC-MS}

• _{Other drugs of abuse group 1 ELISA}

(4)

Why switch to LC-MS method?

Benefits of replacing GC-MS

• Faster less need for chromatographic separation

• Less sample prep no derivatization

• No thermal instability benzodiazepines analysis

• No volatility limitations

Benefits of replacing immunoassay

• Lower consumables cost

• More specific

(5)

Goal

• Develop fast, easy to use, generic LC-MS method to analyze

multiple classes of drugs of abuse in urine

• Method has to meet industry standards for

• Precision

• Accuracy

• Limit of quantitation

• Robustness

• With simplest sample prep procedure

Opiates

Benzodiazepines

6-MAM

Other drugs of

abuse group1

Other drugs of abuse

group 2

(6)

Exactive uHRAM LC-MS system

• Resolution

• 100,000 at 1 scan per sec

• 10,000 at 10 scans per sec

• Mass accuracy

• Sub ppm

• Scan speed

• Up to 10 scans per second

•

• Mass range

• m/z 50 - 4000

(7)

Power of mass resolution – Ethinyl Estradiol

Resolution:

10k

,

30k

,

50k

,

100k

279.12

279.14 m/z

279.16

279.18

279.20

0

10

20

30

40

50

60

70

80

90

100 R

e

la

ti

v

e

A

b

u

n

d

a

n

c

e

Ethinyl-Estradiol, 279.17434

Butyl-Phthalate, 279.15909

(8)

(9)

(10)

Exactive quantitative analysis workflow

Sample Preparation

SPE or urine dilution

Data processing

extract chromatograms with 2 ppm mass accuracy

Custom reports

LC-MS method

(11)

Urine sample preparation

SPE

Int. std addition

Enzymatic Hydrolysis

SPE Extraction

Evaporation

Reconstitution

Urine dilution

Int. std addition

20 X dilution with 25-50% MeOH

Inject Samples

Benzodiazepines, opiates and

Other drugs of abuse group 2

Other drugs of abuse

(12)

Blood sample preparation

SPE

Int. std addition

SPE Extraction

Evaporation

Reconstitution

Benzodiazepines, opiates

other drugs of abuse group 2

Precipitation/dilution

Int. std addition

ACN addition in ratio 1:1

Centrifugation

30 X dilution

Other drugs of abuse

group 1

(13)

LC method

• 150 x 2.1 mm, 5 um PFP column

• Mobile phase

• A: 20 mm NH4Ac, 0.1% FA in DIW

• B: 0.1% FA in ACN

• 7 min gradient

(14)

MS method

• HESI source

• Full scan data in compounds specific m/z range

• Resolution 100K

(15)

Opiates and Opioids in Urine

Hydrocodone

Codeine

Morphine

Hydromorphone

Oxycodone

dihydrocodeine

Normeperidine

Oxymorphine

Meperidine

Oxymorphone D3

Oxycodone D6

Dihydrocodeine D6

Meperidine D6

Hydrocodone D6

Hydromorphone D6

Codeine D6

Morphine D6

(16)

Hydrocodone Y = 0.0744648+0.000852152*X R^2 = 0.9401 W: 1/X^2 0 2000 4000 6000 8000 10000 12000 ng/mL 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 A re a R a ti o Codeine Y = 0.0702431+0.000851922*X R^2 = 0.9461 W: 1/X^2 0 2000 4000 6000 8000 10000 ng/mL 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 A re a R a tio Meperidine Y = 0.075201+0.000849812*X R^2 = 0.9540 W: 1/X^2 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 A re a Ra ti o Morphine Y = 0.054877+0.000912426*X R^2 = 0.9288 W: 1/X^2 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 A re a R a ti o

Hydrocodone

Codeine

Meperidine

Morphine

Opiates and Opioids in Urine - dynamic range

(17)

Oxymorphone Y = 0.0572347+0.000794734*X R^2 = 0.9411 W: 1/X^2 0 2000 4000 6000 8000 10000 ng/mL 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 A re a R a ti o Normeperidine Y = 0.0229096+0.00023572*X R^2 = 0.9434 W: 1/X^2 0 2000 4000 6000 8000 10000 ng/mL 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 A re a R a ti o Oxymorphone Y = 0.0572347+0.000794734*X R^2 = 0.9411 W: 1/X^2 0 2000 4000 6000 8000 10000 ng/mL 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 A re a R a ti o Oxycodone Y = 0.0712176+0.000849364*X R^2 = 0.9510 W: 1/X^2 0 2000 4000 6000 8000 10000 ng/mL 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 A re a R a ti o

Normeperidine

Oxymorphone

Oxycodone

Dihydrocodeine

Opiates and Opioids in Urine- dynamic range

(18)

GC-MS correlation

No

Analyte

Exactive LC-MS

(ng/mL)

GC-MS

(ng/mL)

1 Morphine

289

343

2 Oxymorphone

117

219

3 Oxycodone

167

166

3 Oxymorphone

121

174

4 Hydrocodone

651

650

4 Hydromorphone

503

552

4 Dihydrocodeine

57

74

5 Oxycodone

1833

1822

5 Oxymorphone

593

574

6 Opiates

0

7 Opiates

0

8 Morphine

22,562

37,954***

8 Hydrocodone

6061

7336

Only

after dilution

(19)

6AMSTD10 - m/z= 328.15-328.15 SM: 5 RT:2.10 - 4.10NL:3.16E5 F: {0,0} + p ESI Full ms [ 327.00-335.00] 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti v e I n te n s it y RT: 3.10 3.40 _3.75

6-MAM 10 ng/mL

6AMSTD10 - m/z= 334.19-334.19 SM: 5 RT:2.10 - 4.10NL:5.70E5 F: {0,0} + p ESI Full ms [ 327.00-335.00] 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti v e I n te n s it y RT: 3.10 3.91 3.41 3.77 4.04

IS 20 ng/mL

6-MAM linearity and dynamic range

6- MAM 3 - 400 ng/mL

6-ACETYLMORPHINE

Y = -0.0899199+0.0613337*X R^2 = 0.9984 W: 1/X

0

50

100

150

200

250

300

350

400 NG/ML

0

2

4

6

8

10

12

14

16

18

20

22

24

26 A

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(20)

STD10 W50000MOR - m/z= 328.15-328.15 SM: 5 RT:

2.11 - 4.11

NL:

4.31E5

F: {0,0} + p ESI Full ms [ 327.00-335.00]

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100 R

e

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n

s

it

y

RT: 3.11

3.32 _3.48

2.86

2.96

3.57

3.69

3.40

2.25 _2.81

2.35 _3.76

2.54

3.87

3.98 Measured =10.6 ng/mL

6-MAM NLCP compliance test

10 ng/mL of 6-MAM

in the presence of 50,000 ng/mL

Morphine

(21)

6-MAM GC-MS Correlation - Samples

No

Exactive LC-MS

(ng/mL)

GC-MS

(ng/mL)

1

16.6

15.4

2

59.9

56.8

3

21.4

20.6

3

18.4

14.6

4

13

10.8

4

7.8

6.6

(22)

Blood samples XIC with mass accuracy of 2 ppm

174876bld - m/z= 308.18-308.18 SM: 5 RT:2.18 - 7.18NL:7.41E5 F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00] 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti ve I n te n s it y RT: 4.68 5.84 5.50 6.63 174876bld - m/z= 314.21-314.21 SM: 5 RT:2.18 - 7.18NL:1.97E6 F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00] 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti ve I n te n s it y RT: 4.68 5.68 5.41 6.22

Zolpidem 293 ng/mL

Zolpidem-D6

1763 72 - m /z= 258 .19-25 8.19 S M: 5 RT:1 .88 - 6.88NL:1.2 8E 6 F : F TM S {0,0} + p E S I F ull m s [ 232.0 0-1000 .00] 50 55 60 65 70 75 80 85 90 95 100 v e I n te n s it y RT: 4.38

Dextrorphan 111 ng/mL

1763 72 - m/z= 261 .20-26 1.20 S M: 5 RT:1 .88 - 6.88NL:1.9 7E 6 F: FTMS {0,0} + p ES I Full ms [ 232.0 0-1000 .00] 50 55 60 65 70 75 80 85 90 95 100 ti v e I n te n s it y RT: 4.38

Dextrorphan-D3

(23)

Blood samples XIC with mass accuracy of 2 ppm

174876bld - m/z= 326.21-326.21 SM: 5 RT:3.34 - 5.34NL:2.18E5 F: {0,0} + p E SI Full ms [ 320.00-1000.00] 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Re la ti v e I n te n s it y RT: 4.34 4.51 4.64 4.79

Norpropoxyphene 168 ng/mL

174876bld - m/z= 331.24-331.24 SM: 5 RT:3.34 - 5.34NL:4.42E5 F: {0,0} + p E SI Full ms [ 320.00-1000.00] 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti v e I n te n s it y RT: 4.34 4.45 4.58 4.70 5.07

Norpropoxyphene-D5

1 74 87 6b ld - m /z= 34 0.23 -3 40 .2 3 S M : 5 RT:3 .3 7 - 5 .3 7NL :7 .5 9E 5 F : {0 ,0 } + p E S I F ull m s [ 32 0.00 -1 00 0.00 ] 3.4 3.6 3 .8 4 .0 4 .2 4 .4 4.6 4.8 5 .0 5 .2 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 10 0 R e la tiv e In te n s it y RT: 4 .3 7 4 .95 4 .81 5.18 3 .9 2 4 .2 2

Propoxyphene 162 ng/mL

S td 1000 ng_mlbld - m /z= 351.30-351.30 S M: 5 RT:3.36 - 5.36NL:4.35E6 F : {0,0} + p E SI F ull ms [ 320.00-1000.00] 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti v e I n te n s it y RT: 4.36 4.71 4.88 5.01 5.15 5.25 3.86 4.06 3.41 3.60 3.73 4.17

Propoxyphene-D5

(24)

154147 - m/z= 220.13-220.13 S M: 5 RT:1.81 - 4.81NL:2.30E6 F: FTMS {0,0} + p ESI Full ms [ 218.00-1000.00] 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R e la ti ve I n te n s it y RT: 3.31 3.71

Ritalinic Acid 293 ng/mL

1541 47 - m /z= 249 .24 -2 49.24 S M: 5 RT:4.30 - 7 .02NL:1.30 E 6 F : F TM S {0,0} + p E S I F ull m s [ 218 .0 0-1 000.00 ] 4.4 4.6 4 .8 5.0 5.2 5 .4 5.6 5.8 6 .0 6.2 6.4 6 .6 6.8 7.0 Tim e (m in) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 10 0 R e la ti v e I n te n s it y RT: 5.80 6.19 6 .5 3 6.84 4 .73 4.9 3 5.12 5.34 6.38 6.69