PO86980511_L_1 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
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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.