Rapid, Simultaneous Screen and Confirmation Analysis Of

C18 Core-shell Column Column Core-shell C18 Fluids byLC/MS/MSUsing a Biological in Benzodiazepines and ConfirmationAnalysisof Screen Simultaneous Rapid, Phenomenex, Inc.,411MadridAve., Torrance, CA 90501USA Liming Peng,andTivadarFarkas

PO86980511_L_1 Introduction

Benzodiazepines, a class of psychoactive drugs known poor crossreactivity, resulting in low sensitivity for detecting for their broad range of therapeutic effects, such as low dosage benzodiazepines in biomatrices1. GC/MS is a sedative-hypnotic, anxiolytic, anesthetic, antidepressant, highly sensitive specific technique capable of accurately anticonvulsant, and anti-insomnia, are among the most identify of target analytes. However, derivatization is commonly prescribed drugs for clinical applications. required prior to analysis by GC/MS since GC is limited to However, as addictive drugs, benzodiazepines have compounds which are volatile at temperatures below 400 the potential for misuse, which can result in impaired °C. In comparison to GC/MS, LC/MS/MS 2,3,4,5 offers superior human performance or fatal cases of drug intoxication. sensitivity, selectivity, and robustness for simultaneously Benzodiazepines are also deliberately misused in sport detecting benzodiazepines and their metabolites in a competition. Consequently, they are classified as controlled complex mixture like biological fluids without any need substances that are included in doping control and routine for derivatization. Furthermore, due to its large dynamic employment screening. The development of adequate and range LC/MS/MS is well accepted as quantitative analytical rapid methods for their screening and confirmation analysis technique. therefore receives a great deal of attention in toxicological, In this work we present a rapid and simultaneous clinical, and forensic laboratories. screening, confirmation, and quantitative analysis of 38 Screening of benzodiazepines is commonly performed by benzodiazepines, non-benzodiazepine hypnotics, and their immunoassay. There are a wide variety of benzodiazepines metabolites in biological fluids - human plasma and urine which have common metabolic pathways and produce - by LC/MS/MS using a C18 core-shell column in a single multiple metabolites. This makes it difficult to develop a chromatographic run taking only 8 minutes. comprehensive and specific immunoassay suitable for all benzodiazepines. Furthermore, some benzodiazepines have Figure 1. Molecular Structures of Benzodiazepines and Metabolites

N O Cl Cl Cl + N Br HO N N O N O Cl N N N N N N N N N N O O H O H Alprazolam Bromazepam Chlordiazepoxide Clobazam Clonazepam

CH3 O O N Cl F + Cl N HO N N Cl O HO N Cl N N

N N N O N N O N O O H Clorazepate Diazepam Demoxepam Estazolam Flunitrazepam

F Cl Cl Cl N Cl Cl Cl Cl N N N N N O N HO HO O N F N N N F O H O F Flurazepam Halazepam Lorazepam Lormetazepam Medazepam

F O Cl

+ N N N O N Cl N Cl N Cl N N N N O Cl N N N O H O Midazolam Nitrazepam Prazepam Triazolam Tetrazepam

N O F N N F + N Cl N O O N O N N N N N N Cl N N HO N O N Cl O N N O O OH OH Zopiclone Zolpidem a-Hydroxyalprazolam 2-Hydroxyethylflurazepam 3-Hydroxyflunitrazepam

O H H F + Cl F N N Cl N O H Cl H N N N N N

N N N N O H O N O H O O H 7-Aminoflunitrazepam N-Desmethylflunitrazepam 7-Aminoclonazepam N-Ethylnordiazepam 4’-Hydroxynordiazepam Figure 2. The Metabolism of Diazepam

CH3 O H O N N

N-dealkylation Cl N Cl N

Diazepam Nordiazepam

3-hydroxylation 3-hydroxylation

CH3 O H O N N OH N-dealkylation OH Cl N Cl N

Temazepam Oxazepam

http://www.toxlab.co.uk/benzodia.htm Figure 3. Specific Pattern of Metabolism of Benzodiazepines

Chlordiazepoxide Clorazepate Medazepam Normedazepam

Demoxepam Ketazolam

3-Hydroxyprazepam Prazepam Nordiazepam Diazepam 4’-Hydroxydiazepam

3-Hydroxyhalazepam Halazepam Oxazepam

4’-Hydroxynordiazepam Temazepam

Lormetazepam Clonazepam Triazolam Alprazolam

Lorazepam 7-Aminoclonazepam α-Hydroxytrizolam α-Hydroxyalprazolam Experimental Conditions

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.

SPE Procedures HPLC Conditions Cartridge: Strata™-X Columns: As Noted Part No.: 8B-S100-TAK Dimensions: As Noted Condition: 1 mL Methanol Mobile Phases: A: 0.1 % Formic acid in Water Equilibrate: 1 mL Water B: 0.1 % Acid in Acetonitrile Load: Load above spiked sample Injection Volume: 2 mL onto SPE cartridge Gradient: 2.6 µm 1.7 µm Wash 1: 1 mL Water Time (min) % B Time (min) % B Wash 2: 1 mL to 15 % Methanol in Water Elute: 1 mL Methanol 0 20 0 20 Dry : Evaporate to dryness 5 70 5 70 Reconstitute : 200 µL Methanol/Water (1:1) 5.5 100 5.1 20 5.51 20 8.0 20 8.5 20 — — 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 Instrumentation HPLC 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 MS Detection Tem. CUR Gas 1 Gas 2 IS CAD 550 ºC 20 psi 55 psi 45 psi 5000 V 4.0 CUR: 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 Figure 4. XIC of 57 Benzodiazepines, Metabolites and Internal Standards Columns: Kinetex® 2.6 µm XB-C18 Dimensions: 100 x 2.1 mm Part No.: 00D-4496-AN Flow Rate: 0.5 mL/min Temperature: 30 °C Backpressure: 382 bar Injection Concentration: 50 ng/mL

2.6e5

2.4e5

2.2e5 s

p 2.0e5 c

, y t i 1.8e5 s n e t n

I 1.6e5

1.4e5

1.2e5

1.0e5

8.0e4

6.0e4

4.0e4

2.0e4 App ID 19763

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min 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-C18 Dimensions: 100 x 2.1 mm Part No.: 00D-4496-AN Flow Rate: 0.5 mL/min Temperature: 35 °C

Backpressure: 307 bar Injection Concentration: 50 ng/mL

4.0e5 In human plasma matrix 3.5e5 3.0e5 2.5e5 2.0e5

Intensity, cps 1.5e5 1.0e5 5.0e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

4.0e5 In urine matrix 3.5e5 3.0e5 2.5e5 2.0e5

Intensity, cps 1.5e5 1.0e5 5.0e4 App ID 19765 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Figure 6. XIC of a Mix of 57 Benzodiazepines, Metabolites and Internal Standards spiked into Human Plasma or Urine (2)

Columns: Kinetex 1.7 µm XB-C18 Dimensions: 50 x 2.1 mm Part No.: 00B-4498-AN Flow Rate: 0.4 mL/min Temperature: 35 °C Backpressure: 253 bar Injection Concentration: 50 ng/mL

3.5e5 In human plasma matrix 3.0e5

2.5e5

2.0e5

1.5e5 Intensity, cps 1.0e5

5.0e4

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

3.5e5 In urine matrix

3.0e5

2.5e5

2.0e5

1.5e5 Intensity, cps 1.0e5

5.0e4 App ID 19766 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min Results and Discussion

LC separation and MS/MS detection for screening In general, drug-protein binding in human plasma is not of identification general concern with all benzodiazepines - only with specific ones. The absolute recoveries of flurazepam, midazolam, Separations of benzodiazepines were carried out on a -hydroxymidazolam, and 7-aminoflunitrazepam from Kinetex 2.6 µm XB-C18 100 x 2.1 mm and a Kinetex 1.7 µm a human plasma were initially very low (<50 %, data not XB-C18 50 x 2.1 mm column in gradient elution mode, using presented). Significant improvement in recoveries of these 0.1 % Formic acid in Water and Acetonitrile as mobile phase. compounds was observed after adding 0.1 M Acetic acid to ESI in positive ion mode, with multiple reaction monitoring plasma samples before SPE with the purpose of disrupting (MRM) and scheduled MRMs were used for screening, analyte-protein binding. confirmation, and quantification of benzodiazepines and their metabolites. Quantification Instead of monitoring all MRM transitions during the entire Method linearity was studied in the concentration range acquisition period, scheduled MRMs monitor only appropriate 1-300 ng/mL (at 1, 5, 10, 50, 100, and 300 ng/mL) with MRM transitions within the expected chromatographic 50 ng/mL of specified deuterated benzodiazepines and elution window. This decreases the number of concurrent metabolites (as internal standard) in human plasma and MRMs monitored at any one time, and allows the use of urine. Calibration curves were constructed based on MRM dwell times and maximized effective duty cycle (analyte peak area ratios (analyte/IS) for the transition of highest utilization). This results in improved accuracy in quantitation intensity for each targeted analyte. The results demonstrate with a larger number of MRM transitions monitored in each good linearity in all cases with R ≥ 0.998 (Table 1). experiment. Recovery and precision The Kinetex 2.6 µm core-shell column provides UHPLC The SPE sample preparation method was evaluated by efficiency at much lower pressure (<385 bar) than columns spiking human plasma or urine with 50 ng/mL benzodiazepine made with sub-2 µm fully porous particles. Due to this lower and their metabolite standards. Absolute analyte recoveries operational pressure, longer core-shell columns can be by SPE-LC/MS/MS were determined by comparing specific used for the highly efficient separation of benzodiazepines peak areas for spiked extracts to same level standards. The in a short analysis time at the relatively higher flow rate of absolute recoveries of benzodiazepines and metabolites 0.4 – 0.5 mL/min ( . Kinetex 2.6 µm XB-C18, Figures 4-6) present in human plasma and urine ranged between 55-115 a newly developed core-shell column, provided equivalent % (Table 1). The precision with SPE-LC/MS/MS analyses is performance to Kinetex 2.6 µm C18 (data not presented), RSD < 15 % for all cases as show in Table 1. but occasionally different selectivity (Figure 4 and Table 1). MRM transitions for specific benzodiazepines All screened benzodiazepines and metabolites could be identified either based on retention times or MRM transitions, Table 1 summarized results on studied benzodiazepines except for desalkylflurazepam/norfludiazepam, which have and metabolites, retention times, parent ions (Q1), and major the same retention and MRM transitions. transition ions (Q3) for quantification and confirmation. SPE offline sample preparation for LC/MS/MS By losing a hydroxyl group, demoxepam provided screening and quantification much stronger signal at the 270.98/192.9 transition than 287.1/241.1. Clorazepate is converted to Benzodiazepines were extracted from human plasma and nordiazepam by losing a carboxylic acid group urine using Strata-X, a mixed mode hydrophilic-hydrophobic (Figure 3). Therefore, clorazepate can be quantified only polymer-based sorbent. The procedure is simple, efficient, based on transitions specific to nordiazepam (270.991/140.1 and reliable, as demonstrated by results shown in and 270.991/165). Thus, it was used for the quantization of . The online SPE sample preparation for screening Table 1 demoxepam instead of 287.1/241.1. and quantification will be explored in the future. Table 1. Summary of Screen and Quantification of Benzodiazepines in Plasma or Urine by LC\MS\MS

c d tR (min) Q1 Q3 (amu) SPE Recovery at 50 ng/mL Linearity Compounds 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

113.3≥1 - Desalkyl furazepam 3.37 3.74 289.0 140.1 226.1 99.09 - 113.39 3.84 - 4.41 1.79 - 3.62 0.9991 0.9993 114.38

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

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

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

12.11 - Tetrazepam 3.00 3.44 289.1 253.0 225.2 55.12 - 78.65 4.74 - 4.99 103.36 - 106.67 0.9989 0.9992 15.18

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

N-Desmethyl 2.91 3.33 304.1 258.1 87.5 7.86 92.62 - 96.80 2.62 - 4.17 flunitrazepam-d4

N-Ethyl 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 nordiazepam

-Hydroxy a 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 midazolam

-Hydroxy a 2.88 3.23 363.1 335.1 87.34 - 109.42 5.18 - 6.23 105.02 - 113.54 3.50 - 4.56 triazolam-d4

-Hydroxy a 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 triazolam-

-Hydroxy a 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 alprazoam

-Hydroxy a 2.86 3.20 330.1 302.1 112.8 7.27 113.83 - 113.85 3.80 - 4.00 alprazolam-d5

2-Hydroxyethyl 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 flurazepam

2-Hydroxyethyl 3.16 3.53 337.0 113.2 100.9 6.35 104.30 - 113.36 5.83 - 4.76 flurazepam-d4

3-Hydroxy 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 flunitrazepam

4’-Hydroxy 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 nordiazepam

7-Amino 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 - clonazepam

7-Amino 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 clonazepam-d4

7-AminoDesmethyl 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 funitrazepam

7-Amino 4.85 5.30 330.1 276.2 96.7 3.99 90.25 - 92.41 1.78 - 1.48 flunitrazepam

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. Results and Discussion (cont.)

• A method for the rapid and simultaneous screen, confirmation, and quantitation of benzodiazepines in biological fluids by LC/MS/MS has been developed and demonstrated.

• Thirty-eight benzodiazepines and metabolites were extracted from urine and plasma using 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.

• Kinetex 2.6 µm core-shell column provides the high efficiency of a sub-2 µm column but with 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.

• The method is well suited for high speed screening and confirmation of benzodiazepines from biological samples in forensic, toxicological, and clinical analysis.

• Online SPE sample preparation for screening and quantification will be explored in the future.