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Rapid, Simultaneous Screen and Confirmation Analysis of Benzodiazepines in Biological Fluids by LC/MS/MS Using a C18 Core-shell Column

Liming Peng, and Tivadar Farkas

Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Benzodiazepines, a class of psychoactive drugs known for their broad range of therapeutic effects, such as sedative-hypnotic, anxiolytic, anesthetic, antidepressant, anticonvulsant, and anti-insomnia, are among the most commonly prescribed drugs for clinical applications. However, as addictive drugs, benzodiazepines have the potential for misuse, which can result in impaired human performance or fatal cases of drug intoxication. Benzodiazepines are also deliberately misused in sport competition. Consequently, they are classified as controlled substances that are included in doping control and routine employment screening. The development of adequate and rapid methods for their screening and confirmation analysis therefore receives a great deal of attention in toxicological, clinical, and forensic laboratories.

Screening of benzodiazepines is commonly performed by immunoassay. There are a wide variety of benzodiazepines which have common metabolic pathways and produce multiple metabolites. This makes it difficult to develop a comprehensive and specific immunoassay suitable for all benzodiazepines. Furthermore, some benzodiazepines have

poor crossreactivity, resulting in low sensitivity for detecting low dosage benzodiazepines in biomatrices1. GC/MS is a highly sensitive specific technique capable of accurately identify of target analytes. However, derivatization is required prior to analysis by GC/MS since GC is limited to compounds which are volatile at temperatures below 400 °C. In comparison to GC/MS, LC/MS/MS 2,3,4,5 offers superior sensitivity, selectivity, and robustness for simultaneously detecting benzodiazepines and their metabolites in a complex mixture like biological fluids without any need for derivatization. Furthermore, due to its large dynamic range LC/MS/MS is well accepted as quantitative analytical technique.

In this work we present a rapid and simultaneous screening, confirmation, and quantitative analysis of 38 benzodiazepines, non-benzodiazepine hypnotics, and their metabolites in biological fluids - human plasma and urine - by LC/MS/MS using a C18 core-shell column in a single chromatographic run taking only 8 minutes.

Introduction

Figure 1. Molecular Structures of Benzodiazepines and Metabolites

N

N

N

N

Cl N

N

N

O

Br

H

N

NCl

OCH3

N

N

ClHO

N

N

N

Cl

O

ON

N

O H

ClN

+

O

O

N

N

O H

Cl

O

OHN

N

ClHO

O

N

N

N

N

ClN

N

O

N+F

O

O

ClN

NO

F

N

N

N

O

FF

F

Cl

N

N

O

Cl

H

Cl

HO

N

N

O

ClCl

HO

N

N

Cl

N

NNCl

F

N

N

Cl

O

N

N

O

N+

H

O

ON

NNCl

Cl

N

N

Cl

O

O

N

N

O

N

N

N

N

Cl

O

N

NN

NCl

OH

N

NN

O

N

N

F

Cl

O

OH

N

N

N+F

OH

O

O

O

N

N

FN

O

H

H

N

N

Cl

O

N

N

Cl

OH

HO

N

N

N+F

O

O

O H

N

N

ClN

HO

H

H

Alprazolam

Midazolam

Clorazepate

Zopiclone

Flurazepam

7-Aminoflunitrazepam

Bromazepam

Nitrazepam

Diazepam

Zolpidem

Halazepam

N-Desmethylflunitrazepam

Chlordiazepoxide

Prazepam

Demoxepam

a-Hydroxyalprazolam

Lorazepam

7-Aminoclonazepam

Clobazam

Triazolam

Estazolam

2-Hydroxyethylflurazepam

Lormetazepam

N-Ethylnordiazepam

Clonazepam

Tetrazepam

Flunitrazepam

3-Hydroxyflunitrazepam

Medazepam

4’-Hydroxynordiazepam

Figure 2. The Metabolism of Diazepam

N

NCl

H ON

NCl

OCH3

N

NCl

OH

OCH3

N

NCl

OH

OH

Diazepam Nordiazepam

Temazepam Oxazepam

N-dealkylation

3-hydroxylation

N-dealkylation

3-hydroxylation

http://www.toxlab.co.uk/benzodia.htm

Figure 3. Specific Pattern of Metabolism of Benzodiazepines

Chlordiazepoxide Clorazepate Medazepam Normedazepam

Ketazolam

Alprazolam

a-Hydroxyalprazolam

Triazolam

a-Hydroxytrizolam

Clonazepam

7-Aminoclonazepam

Lormetazepam

Lorazepam

Prazepam

3-Hydroxyhalazepam

Nordiazepam3-Hydroxyprazepam

Halazepam

4’-Hydroxynordiazepam

Diazepam

Oxazepam

Temazepam

4’-Hydroxydiazepam

Demoxepam

Experimental Conditions

HPLC ConditionsColumns: As Noted

Dimensions: As NotedMobile Phases: A: 0.1 % Formic acid in Water

B: 0.1 % Acid in AcetonitrileInjection Volume: 2 mL

Gradient: 2.6 µm 1.7 µm

Time (min) % B Time (min) % B

0 20 0 20

5 70 5 70

5.5 100 5.1 20

5.51 20 8.0 20

8.5 20 — —

SPE ProceduresCartridge: Strata™-X

Part No.: 8B-S100-TAKCondition: 1 mL Methanol

Equilibrate: 1 mL WaterLoad: Load above spiked sample

onto SPE cartridgeWash 1: 1 mL WaterWash 2: 1 mL to 15 % Methanol in Water

Elute: 1 mL MethanolDry : Evaporate to dryness

Reconstitute : 200 µL Methanol/Water (1:1)

Benzodiazepines and metabolites were spiked into 200 µL of urine or human plasms at different concentration levels, along with 50 ng/mL internal standards (IS). To the plasma samples, 100 µL of 1M acetic acid was added and the sample was diluted to 1 mL with water.

MS DetectionTem. CUR Gas 1 Gas 2 IS CAD

550 ºC 20 psi 55 psi 45 psi 5000 V 4.0CUR: curtain gas; Gas 1: nebulizer gas; Gas 2: turbo gas; IS: IonSpray voltage; CAD: Collision Gas

Scheduled MRM: MRM detection window: 20 s; Target scan time: 1

Sample:

1. Alprazolam 11. Flunitrazepam 21. Oxazepam 31. 2-Hydroxyethylflurazepam

2. Bromazepam 12. Flurazepam 22. Prazepam 32. 3-Hydroxyflunitrazepam

3. Chlordiazepoxide 13. Halazepam 23. Temazepam 33. 4’-Hydroxynordiazepam

4. Clobazam 14. Lorazepam 24. Triazolam 34. 7-Aminodesmethylflunitrazepam

5. Clonazepam 15. Lormetazepam 25. Tetrazepam 35. 7-Aminoclonazepam

6. Clorazepate 16. Medazepam 26. Zopiclone 36. 7-Aminoflunitrazepam

7. Diazepam 17. Midazolam 27. Zolpidem 37. N-Desmethylflunitrazepam

8. Demoxepam 18. Nitrazepam 28. a-Hydroxyalprazolam 38. N-Ethylnordiazepam

9. Desalkylflurazepam 19. Nordiazepam 29. a-Hydroxymidazolam

10. Estazolam 20. Norfludiazepam 30. a-Hydroxytriazolam

InstrumentationHPLC System: Agilent 1200 SL

Pump: G1312B (Binary Pump)Autosampler: G1337C HP-ALS-SL MS Detector: AB Sciex API4000™ LC/MS/MS Turbo V™ Source with ESI probe

Figure 4. XIC of 57 Benzodiazepines, Metabolites and Internal Standards

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

Inte

nsit

y, c

ps

Ap

p ID

197

63

Columns: Kinetex® 2.6 µm XB-C18Dimensions: 100 x 2.1 mm

Part No.: 00D-4496-ANFlow Rate: 0.5 mL/min

Temperature: 30 °CBackpressure: 382 bar

Injection Concentration: 50 ng/mL

Figure 5. XIC of a Mix of 57 Benzodiazepines, Metabolites and Internal Standards spiked into Human Plasma or Urine (1)

Columns: Kinetex 2.6 µm XB-C18Dimensions: 100 x 2.1 mm

Part No.: 00D-4496-ANFlow Rate: 0.5 mL/min

Temperature: 35 °CBackpressure: 307 bar

Injection Concentration: 50 ng/mL

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.50.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Inte

nsity

, cp

sIn

tens

ity, c

ps

5.0 5.50.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

Ap

p ID

197

65

In human plasma matrix

In urine matrix

Figure 6. XIC of a Mix of 57 Benzodiazepines, Metabolites and Internal Standards spiked into Human Plasma or Urine (2)

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

Inte

nsi

ty,

cp

sIn

ten

sity

, c

ps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.00.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

Ap

p ID

197

66

In human plasma matrix

In urine matrix

Columns: Kinetex 1.7 µm XB-C18Dimensions: 50 x 2.1 mm

Part No.: 00B-4498-ANFlow Rate: 0.4 mL/min

Temperature: 35 °CBackpressure: 253 bar

Injection Concentration: 50 ng/mL

LC separation and MS/MS detection for screening identification

Separations of benzodiazepines were carried out on a Kinetex 2.6 µm XB-C18 100 x 2.1 mm and a Kinetex 1.7 µm XB-C18 50 x 2.1 mm column in gradient elution mode, using 0.1 % Formic acid in Water and Acetonitrile as mobile phase. ESI in positive ion mode, with multiple reaction monitoring (MRM) and scheduled MRMs were used for screening, confirmation, and quantification of benzodiazepines and their metabolites.

Instead of monitoring all MRM transitions during the entire acquisition period, scheduled MRMs monitor only appropriate MRM transitions within the expected chromatographic elution window. This decreases the number of concurrent MRMs monitored at any one time, and allows the use of dwell times and maximized effective duty cycle (analyte utilization). This results in improved accuracy in quantitation with a larger number of MRM transitions monitored in each experiment.

The Kinetex 2.6 µm core-shell column provides UHPLC efficiency at much lower pressure (<385 bar) than columns made with sub-2 µm fully porous particles. Due to this lower operational pressure, longer core-shell columns can be used for the highly efficient separation of benzodiazepines in a short analysis time at the relatively higher flow rate of 0.4 – 0.5 mL/min (Figures 4-6). Kinetex 2.6 µm XB-C18, a newly developed core-shell column, provided equivalent performance to Kinetex 2.6 µm C18 (data not presented), but occasionally different selectivity (Figure 4 and Table 1).

All screened benzodiazepines and metabolites could be identified either based on retention times or MRM transitions, except for desalkylflurazepam/norfludiazepam, which have the same retention and MRM transitions.

SPE offline sample preparation for LC/MS/MS screening and quantification

Benzodiazepines were extracted from human plasma and urine using Strata-X, a mixed mode hydrophilic-hydrophobic polymer-based sorbent. The procedure is simple, efficient, and reliable, as demonstrated by results shown in Table 1. The online SPE sample preparation for screening and quantification will be explored in the future.

In general, drug-protein binding in human plasma is not of general concern with all benzodiazepines - only with specific ones. The absolute recoveries of flurazepam, midazolam, a-hydroxymidazolam, and 7-aminoflunitrazepam from human plasma were initially very low (<50 %, data not presented). Significant improvement in recoveries of these compounds was observed after adding 0.1 M Acetic acid to plasma samples before SPE with the purpose of disrupting analyte-protein binding.

Quantification

Method linearity was studied in the concentration range 1-300 ng/mL (at 1, 5, 10, 50, 100, and 300 ng/mL) with 50 ng/mL of specified deuterated benzodiazepines and metabolites (as internal standard) in human plasma and urine. Calibration curves were constructed based on MRM peak area ratios (analyte/IS) for the transition of highest intensity for each targeted analyte. The results demonstrate good linearity in all cases with R ≥ 0.998 (Table 1).

Recovery and precision

The SPE sample preparation method was evaluated by spiking human plasma or urine with 50 ng/mL benzodiazepine and their metabolite standards. Absolute analyte recoveries by SPE-LC/MS/MS were determined by comparing specific peak areas for spiked extracts to same level standards. The absolute recoveries of benzodiazepines and metabolites present in human plasma and urine ranged between 55-115 % (Table 1). The precision with SPE-LC/MS/MS analyses is RSD < 15 % for all cases as show in Table 1.

MRM transitions for specific benzodiazepines

Table 1 summarized results on studied benzodiazepines and metabolites, retention times, parent ions (Q1), and major transition ions (Q3) for quantification and confirmation.

By losing a hydroxyl group, demoxepam provided much stronger signal at the 270.98/192.9 transition than 287.1/241.1. Clorazepate is converted to nordiazepam by losing a carboxylic acid group (Figure 3). Therefore, clorazepate can be quantified only based on transitions specific to nordiazepam (270.991/140.1 and 270.991/165). Thus, it was used for the quantization of demoxepam instead of 287.1/241.1.

Results and Discussion

Table 1. Summary of Screen and Quantification of Benzodiazepines in Plasma or Urine by LC\MS\MS

Norfludiazepam 3.36 3.75 288.9 140.1 226.2 92.69 - 98.24 5.10 - 6.81 99.49 - 109.72 6.40 - 12.82 0.9990 0.9992

Oxazepam 3.01 3.38 287.0 241.2 268.9 96.60 - 101.64 4.39 - 5.56 97.22 - 104.91 3.61 - 7.28 0.9989 0.9997

Prazepam 4.87 5.33 325.0 271.1 140.0 96.7 7.55 83.60 - 86.88 3.93 - 4.95 0.9995 0.9992

Prazepam-d5 4.85 5.30 330.1 276.2 96.7 3.99 90.25 - 92.41 1.78 - 1.48

Temazepam 3.50 3.90 301.1 255.2 257.2 90.12 - 108.95 4.07 - 6.01 97.83 - 112.78 4.89 - 9.25 0.9991 0.9993

Temazepam-d5 3.48 3.88 306.1 260.2 91.89 - 109.28 4.57 - 4.80 101.28 -111.85 2.43 - 2.63

Tetrazepam 3.00 3.44 289.1 253.0 225.2 55.12 - 78.65 4.74 - 4.99 103.36 - 106.6712.11 - 15.18

0.9989 0.9992

Triazolam 3.32 3.67 343.0 315.2 238.9 71.01 - 104.67 5.48 - 9.93 117.5 - 113.92 6.73 - 2.19 0.9994 0.9997

Triazolam-d4 3.32 3.88 347.0 312.2 75.18 - 104.76 7.40 - 8.94 112.49 - 113.94 4.99 - 5.03

Zolpidem 0.81 1.34 308.2 235.2 236.2 97.64 - 100.44 2.71 - 3.89 83.09 - 90.85 1.83 - 3.96 0.9995 0.9994

Zolpidem-d6 0.79 1.31 314.2 235.2 95.8 3.1 89.13 - 92.86 2.63 - 3.71

Zopiclone 0.53 0.85 389.1 245.1 217.0 78.3 3.28 111.5 14.6 0.9997 0.9997

Zopiclone-d4 0.53 0.84 393.0 245.2 57.1 6.51 105.1 14.6

N-Desmethyl flunitrazepam

2.93 3.35 300.1 254.1 198.1 88.30 - 104.41 4.22 - 7.70 93.16 - 94.37 2.48 - 5.46 0.9997 0.9997

Estazolam 3.02 3.37 295.1 205.2 267.1 96.71 - 112.03 2.59 - 5.13 107.71 - 111.11 4.41 - 5.46 0.9990 0.9997

Estazolam-d5 3.00 3.35 300.1 272.2 98.29 - 113.19 5.30 -12.50 107.43 - 109.75 1.34 - 4.25

Flurazepam 1.93 2.27 388.0 315.1 288.2 71.7 8.1 71.95 - 86.46 6.05 - 6.45 0.9980 0.9997

Flunitrazepam 3.43 3.85 314.1 268.2 239.1 66.03 - 92.22 3.84 - 10.88 95.69 - 103.51 6.09 - 10.45 0.9994 0.9992

Flunitrazepam-d7 3.40 3.82 321.0 275.3 65.84 - 102.31 3.90 - 13.25 106.42 - 112.67 1.80 - 12.43

Halazepam 4.93 5.38 353.0 241.2 222.1 89.32 - 90.71 6.31 - 7.89 87.15 - 91.72 4.62 - 6.78 0.9996 0.9991

Lorazepam 3.40 3.82 321.4 275.1 277.0 74.29 - 106.21 1.68 - 5.81 104.65 - 109.83 5.10 - 5.44 0.9988 0.9995

Lorazepam-d4 3.15 3.52 325.0 279.1 102.78 - 102.57 4.96 - 5.05 104.97 - 112.11 2.67 - 5.50

Lormetazepam 3.71 4.11 335.0 289.1 291.1 98.13 - 109.38 4.92 - 5.45 95.72 - 108.35 8.82 - 10.94 0.9988 0.9997

Medazepam 1.76 2.18 271.1 91.1 207.3 65.30 - 65.75 1.58 - 10.58 74.87 - 83.04 3.13 - 7.69 0.9991 0.9993

Midazolam 1.74 2.15 326.1 291.2 222.0 72.4 11.2 56.60 - 62.18 4.05 - 6.46 0.9989 0.9992

Nitrazepam 2.88 3.30 282.1 180.2 236.1 84.30 - 94.64 3.32 - 7.30 101.45 - 109.51 6.94 - 10.27 0.9999 0.9980

Nordiazepam 3.07 3.49 271.2 140.2 164.9 92.89 - 96.17 3.50 - 5.89 99.21 - 108.43 4.86 - 9.31 0.9995 0.9991

Nordiazepam-d5 3.45 3.45 276.1 140.1 86.25 - 90.91 3.18 - 11.03 108.77 - 108.95 2.42 - 5.09

CompoundstR (min) Q1 Q3 (amu) SPE Recoveryc at 50 ng/mL Linearityd

1.7 µma 2.6 µmb (amu) quantifer qualifier Plasma RSD% Urine RSD% Plasma Urine

Alprazepam 3.20 3.53 309.1 281.2 205.1 100.04 - 109.90 2.50 - 4.67 110.17 - 115.42 2.48 - 2.96 0.9996 0.9997

Alprazolam-d5 3.18 3.51 314.1 286.1 104.17 - 100.57 4.64 - 14.80 113.11 - 115.75 1.23 - 3.79

Bromazepam 2.09 2.50 316.0 182.1 209.2 111.55 - 106.5 2.57 - 9.71 102.83 - 105.15 3.41 - 6.99 0.9992 0.9997

Chlordiazepoxide 1.03 1.60 300.1 227.1 283.2 98.31 - 106.17 3.56 - 9.28 85.23 - 89.56 2.42 - 4.06 0.9995 0.9995

Clobazam 3.64 4.05 301.1 259.1 224.3 99.90 - 10.00 2.79 - 6.23 97.13 - 112.50 4.41 - 4.70 0.9990 0.9989

Clonazepam 3.19 3.58 316.0 270.1 214.0 98.77 - 106.48 1.32 - 6.25 94.97 - 102.61 2.61 - 4.80 0.9990 0.9998

Clonazepam-d4 3.17 3.56 320.1 274.1 102.85 - 105.84 5.44 - 9.56 91.96 - 106.38 2.93 - 3.24

Clorazepate 3.08 3.48 271.0 140.1 165.0 113.5 5.65 105.51 - 110.63 2.90 - 5.08 0.9993 0.9995

Demoxepam 3.52 3.88 271.0 192.9 287/180 113.2 7.98 99.85 - 103.27 1.29 - 6.54 0.9995 0.9995

Demoxepam-d5 2.99 3.37 292.0 246.1 109.5 6.63 86.53 - 97.72 1.49 - 7.61

Desalkyl furazepam 3.37 3.74 289.0 140.1 226.1 99.09 - 113.39 3.84 - 4.41113.3≥1 -

114.381.79 - 3.62 0.9991 0.9993

Desalkyl flurazepam-d4 3.16 3.72 293.0 140.0 86.32 - 112.89 4.14 - 9.41 111.61 - 114.52 3.30 - 5.03

Diazepam 3.79 4.21 285.1 154.1 193.2 88.22 - 102.46 2.61 - 5.08 83.53 - 96.77 2.95 - 5.23 0.9993 0.9995

Diazepam-d5 3.75 4.17 290.1 154.0 86.32 - 98.53 1.74 - 9.41 96.13 - 99.05 4.87 - 5.56

N-Desmethyl flunitrazepam-d4

2.91 3.33 304.1 258.1 87.5 7.86 92.62 - 96.80 2.62 - 4.17

N-Ethyl nordiazepam

4.33 4.75 299.1 271.1 242.1 87.22 - 102.48 3.46 - 5.60 88.42 - 91.43 4.06 - 4.90 0.9996 0.9999

a-Hydroxy midazolam

1.73 2.15 342.0 324.1 203.1 89.8 2.8 92.66 - 114.91 3.78 - 3.90 0.9992 0.9992

a-Hydroxy triazolam-d4

2.88 3.23 363.1 335.1 87.34 - 109.42 5.18 - 6.23 105.02 - 113.54 3.50 - 4.56

a-Hydroxy triazolam-

2.90 3.23 358.9 331.1 176.0 89.83 - 114.87 2.33 - 7.50 110.36 - 113.65 3.43 - 3.68 0.9991 0.9993

a-Hydroxy alprazoam

2.87 3.22 325.1 216.2 204.9 104.56 - 113.21 3.52 - 5.78 113.17-110.81 3.23 - 6.77 0.9996 0.9994

a-Hydroxy alprazolam-d5

2.86 3.20 330.1 302.1 112.8 7.27 113.83 - 113.85 3.80 - 4.00

2-Hydroxyethyl flurazepam

3.18 3.55 333.0 109.1 211.2 96.46 - 105.62 2.0 - 7.92 111.58 - 112.08 8.73 - 3.86 0.9996 0.9993

2-Hydroxyethyl flurazepam-d4

3.16 3.53 337.0 113.2 100.9 6.35 104.30 - 113.36 5.83 - 4.76

3-Hydroxy flunitrazepam

3.18 3.32 330.1 284.2 85.29 - 98.37 6.03 - 12.22 109.24 - 111.29 2.93 - 3.39 0.9993 0.9998

4’-Hydroxy nordiazepam

0.82 1.31 287.0 165.0 99.16 - 105.78 1.67 - 4.47 93.53 - 103.47 4.76 - 5.19 0.9990 0.9994

7-Amino - clonazepam

3.36 3.75 288.9 140.1 226.2 92.69 - 98.24 5.10 - 6.81 99.49 - 109.72 6.40 - 12.82 0.9990 0.9992

7-Amino clonazepam-d4

3.01 3.38 287.0 241.2 268.9 96.60 - 101.64 4.39 - 5.56 97.22 - 104.91 3.61 - 7.28 0.9989 0.9997

7-AminoDesmethyl funitrazepam

4.87 5.33 325.0 271.1 140.0 96.7 7.55 83.60 - 86.88 3.93 - 4.95 0.9995 0.9992

7-Amino flunitrazepam

4.85 5.30 330.1 276.2 96.7 3.99 90.25 - 92.41 1.78 - 1.48

a. Kinetex 1.7 µm XB-C18 50 x 2.1 mm

b. Kinetex 2.6 µm XB-C18 100 x 2.1 mm

c. Inter-assay (n=6)

d. Concentration range: 1-300 ng/mL by spiking 5 pts (1, 5, 10, 50, 100, 300 ng/mL) into human plasma or urine, one set.

Table 1. Summary of Screen and Quantification of Benzodiazepines in Plasma or Urine by LC\MS\MS (cont)

LC separation and MS/MS detection for screening identification

Separations of benzodiazepines were carried out on a Kinetex 2.6 µm XB-C18 100 x 2.1 mm and a Kinetex 1.7 µm XB-C18 50 x 2.1 mm column in gradient elution mode, using 0.1 % Formic acid in Water and Acetonitrile as mobile phase. ESI in positive ion mode, with multiple reaction monitoring (MRM) and scheduled MRMs were used for screening, confirmation, and quantification of benzodiazepines and their metabolites.

Instead of monitoring all MRM transitions during the entire acquisition period, scheduled MRMs monitor only appropriate MRM transitions within the expected chromatographic elution window. This decreases the number of concurrent MRMs monitored at any one time, and allows the use of dwell times and maximized effective duty cycle (analyte utilization). This results in improved accuracy in quantitation with a larger number of MRM transitions monitored in each experiment.

The Kinetex 2.6 µm core-shell column provides UHPLC efficiency at much lower pressure (<385 bar) than columns made with sub-2 µm fully porous particles. Due to this lower operational pressure, longer core-shell columns can be used for the highly efficient separation of benzodiazepines in a short analysis time at the relatively higher flow rate of 0.4 – 0.5 mL/min (Figures 4-6). Kinetex 2.6 µm XB-C18, a newly developed core-shell column, provided equivalent performance to Kinetex 2.6 µm C18 (data not presented), but occasionally different selectivity (Figure 4 and Table 1).

All screened benzodiazepines and metabolites could be identified either based on retention times or MRM transitions, except for desalkylflurazepam/norfludiazepam, which have the same retention and MRM transitions.

SPE offline sample preparation for LC/MS/MS screening and quantification

Benzodiazepines were extracted from human plasma and urine using Strata-X, a mixed mode hydrophilic-hydrophobic polymer-based sorbent. The procedure is simple, efficient, and reliable, as demonstrated by results shown in Table 1. The online SPE sample preparation for screening and quantification will be explored in the future.

In general, drug-protein binding in human plasma is not of general concern with all benzodiazepines - only with specific ones. The absolute recoveries of flurazepam, midazolam, a-hydroxymidazolam, and 7-aminoflunitrazepam from human plasma were initially very low (<50 %, data not presented). Significant improvement in recoveries of these compounds was observed after adding 0.1 M Acetic acid to plasma samples before SPE with the purpose of disrupting analyte-protein binding.

Quantification

Method linearity was studied in the concentration range 1-300 ng/mL (at 1, 5, 10, 50, 100, and 300 ng/mL) with 50 ng/mL of specified deuterated benzodiazepines and metabolites (as internal standard) in human plasma and urine. Calibration curves were constructed based on MRM peak area ratios (analyte/IS) for the transition of highest intensity for each targeted analyte. The results demonstrate good linearity in all cases with R ≥ 0.998 (Table 1).

Recovery and precision

The SPE sample preparation method was evaluated by spiking human plasma or urine with 50 ng/mL benzodiazepine and their metabolite standards. Absolute analyte recoveries by SPE-LC/MS/MS were determined by comparing specific peak areas for spiked extracts to same level standards. The absolute recoveries of benzodiazepines and metabolites present in human plasma and urine ranged between 55-115 % (Table 1). The precision with SPE-LC/MS/MS analyses is RSD < 15 % for all cases as show in Table 1.

MRM transitions for specific benzodiazepines

Table 1 summarized results on studied benzodiazepines and metabolites, retention times, parent ions (Q1), and major transition ions (Q3) for quantification and confirmation.

By losing a hydroxyl group, demoxepam provided much stronger signal at the 270.98/192.9 transition than 287.1/241.1. Clorazepate is converted to nordiazepam by losing a carboxylic acid group (Figure 3). Therefore, clorazepate can be quantified only based on transitions specific to nordiazepam (270.991/140.1 and 270.991/165). Thus, it was used for the quantization of demoxepam instead of 287.1/241.1.

Results and Discussion (cont.)

Conclusions

• Amethodfortherapidandsimultaneousscreen,confirmation,andquantitationof benzodiazepines in biological fluids by LC/MS/MS has been developed and demonstrated.

• Thirty-eightbenzodiazepinesandmetaboliteswereextractedfromurineandplasmausing Strata-X SPE with simple wash steps; HPLC separation was carried out using a Kinetex XB-C18 core-shell column with a gradient elution. 37 of the screened benzodiazepines and metabolites were identified based on retention times or MRM transitions.

• Kinetex2.6µmcore-shellcolumnprovidesthehighefficiencyofasub-2µmcolumnbutwith much lower backpressure. This allows the use of longer core-shell columns, such as 100 x 2.1 mm packed with 2.6 µm particles, with relatively higher flow rates and very short analysis times.

• Themethodiswellsuitedforhighspeedscreeningandconfirmationofbenzodiazepinesfrom biological samples in forensic, toxicological, and clinical analysis.

• OnlineSPEsamplepreparationforscreeningandquantificationwillbeexploredinthefuture.