Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. CSF Collection
2.3. Sample Treatments
2.4. LC-MS/MS
2.5. Validation Parameters
2.5.1. Interferences Studies
2.5.2. Limit of Detection (LOD) and Lower Limit of Quantification (LLOQ)
2.5.3. Calibration Model
2.5.4. Bias and Precision
2.5.5. Recovery Rate (RR), Matrix Effect (ME) and Carry-Over
3. Results
3.1. MRM Transitions and Chromatographic Separation
3.2. Sample Treatment Procedure
3.3. Methods Validation
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound | Ion Fragments (m/z) | Collision Energies (V) | Retention Time (min) |
---|---|---|---|
BDZ/Antidepressants | |||
7-aminoclonazepam | 286: 222, 121 | 25, 30 | 7.1 |
7-aminoflunitrazepam | 284: 227, 135 | 25, 28 | 8.5 |
7-aminonitrazepam | 252: 208, 121 | 35, 29 | 3.9 |
alprazolam | 309: 281, 205 | 40, 25 | 12.2 |
bromazepam | 316: 209, 182 | 25, 33 | 10.6 |
brotizolam | 393: 314, 279 | 21, 29 | 11.6 |
clonazepam | 316: 270, 214 | 25, 40 | 11.4 |
chlordiazepoxide | 300: 283, 282 | 9, 21 | 9.5 |
delorazepam | 305: 165, 162 | 29, 33 | 11.7 |
diazepam | 285: 193, 154 | 33, 25 | 11.8 |
flunitrazepam | 314: 268, 239 | 25, 37 | 11.6 |
flurazepam | 388: 317, 315 | 17, 25 | 10.7 |
lorazepam | 321: 303, 275 | 13, 17 | 11.4 |
lormetazepam | 335: 317, 289 | 9, 17 | 11.8 |
midazolam | 326: 291, 223 | 25, 40 | 10.4 |
nordiazepam | 271: 165, 155 | 29, 29 | 1.6 |
oxazepam | 287: 269, 241 | 9, 21 | 11.3 |
pinazepam | 309: 269, 241 | 29, 33 | 12.2 |
prazepam | 325: 271, 140 | 21, 40 | 12.4 |
temazepam | 301: 283, 255 | 9, 21 | 11.6 |
triazolam | 343: 315, 308 | 29, 25 | 11.5 |
α-hydroxyalprazolam | 325: 297, 216 | 30, 35 | 12.4 |
α-hydroxyflunitrazepam | 330: 311, 284 | 35, 30 | 11.8 |
α-hydroxymidazolam | 342: 203, 168 | 35, 45 | 10.6 |
Main drugs of abuse | |||
6-MAM | 328: 211, 165 | 25, 40 | 5.1 |
codeine | 300: 165, 58 | 40, 33 | 3.7 |
DXM | 272: 171, 147 | 42, 30 | 10.2 |
methadone | 310: 265, 105 | 13, 29 | 11.2 |
morphine | 286: 165, 155 | 40, 37 | 1.6 |
oxycodone | 316: 298, 256 | 17, 25 | 4.5 |
tramadol | 264: 246, 58 | 25, 30 | 7.7 |
Others | |||
MDA | 180: 163,105 | 9, 25 | 4.1 |
MDMA | 194: 163, 105 | 9, 25 | 4.6 |
methamphetamine | 150: 119, 91 | 21, 9 | 5.1 |
MDPV | 276: 135, 126 | 25, 25 | 8.2 |
ketamine | 238: 179, 125 | 13, 29 | 6.5 |
norketamine | 224: 207, 125 | 9, 25 | 6.2 |
cocaine | 304: 182, 82 | 17, 33 | 8.3 |
benzoylecgonine | 290: 168, 105 | 17, 29 | 6.8 |
Compound | LOD (ng/mL) | LOQ (ng/mL) | R2 | %MRE | %CV | ME (%) | RR (%) | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
QC1 * | QC2 | QC3 | QC1 | QC2 | QC3 | ||||||
6-MAM | 0.01 | 0.05 | 0.9901 | 10 | 6 | −20 | 13 | 12 | 5.5 | −12 | 88 |
7-aminoclonazepam 1 | 2 | 5 | 0.9877 | 13 | 2 | −18 | 18 | 8 | 7.5 | −15 | 87 |
7-aminoflunitrazepam | 0.03 | 0.05 | 0.9900 | 3 | 2 | 1 | 3.7 | 5 | 1.3 | −20 | 71 |
7-aminonitrazepam 2 | 0.1 | 0.5 | 0.9899 | −1 | 5 | 3 | 2 | 8 | 5 | −25 | 61 |
alprazolam 2 | 0.01 | 0.5 | 0.9989 | −4 | 2 | −4 | 5.2 | 7 | 5.2 | −18 | 71 |
benzoylecgonine | 0.01 | 0.05 | 0.9991 | 9 | 1 | −1 | 13.3 | 10 | 1.5 | −8.5 | 75 |
bromazepam 1 | 1 | 5 | 0.9919 | −1 | 7 | 4 | 1.7 | 8 | 5.3 | −25 | 62 |
brotizolam | 0.03 | 0.1 | 0.9875 | 13 | 10 | 1 | 18 | 16 | 1 | −15 | 65 |
clonazepam 1 | 1 | 5 | 0.9989 | −41 | −10 | −25 | 59 | 18 | 6 | −33 | 65 |
delorazepam | 0.01 | 0.05 | 0.9940 | 3 | 1 | 1 | 5 | 1.7 | 0.4 | −18 | 75 |
chlordiazepoxide 2 | 0.3 | 0.5 | 0.9904 | 2 | 9 | 1 | 3.2 | 7 | 1.1 | −30 | 68 |
cocaine | 0.01 | 0.05 | 0.9859 | 5 | 1 | 0 | 7 | 10 | 0.3 | −6 | 72 |
codeine 2 | 0.1 | 0.05 | 0.9964 | 12 | 10 | −18 | 15 | 12 | 10 | 16 | 70 |
DXM | 0.02 | 0.05 | 0.9967 | −13 | −1 | 4 | 18 | 4 | 4 | −4 | 76 |
diazepam | 0.03 | 0.05 | 0.9999 | −12 | 5 | 1 | 16 | 16 | 2 | −9 | 83 |
flunitrazepam | 0.01 | 0.05 | 0.9911 | −4 | −7 | 1 | 1 | 6 | 0.4 | −32 | 66 |
flurazepam | 0.01 | 0.05 | 0.9871 | 23 | −7 | 15 | 10 | 12 | 1.8 | −31 | 59 |
ketamine | 0.01 | 0.05 | 0.9967 | −5 | 5 | 2 | 1 | 1 | 3 | −2 | 83 |
lorazepam 2 | 0.1 | 0.5 | 0.9999 | 2 | 7 | 1 | 19 | 20 | 0.4 | −33 | 64 |
lormetazepam 2 | 0.2 | 0.5 | 0.9979 | 20 | 9 | 19 | 14 | 6.8 | 1.8 | −37 | 67 |
MDA 2 | 0.01 | 0.05 | 0.9981 | 2 | 12 | 15 | 8 | 5 | 2 | 10 | 81 |
MDMA | 0.01 | 0.05 | 0.9999 | 10 | 3 | 6 | 2 | 4 | 3 | −8 | 75 |
MDPV | 0.01 | 0.05 | 0.9956 | 2 | 2 | 11 | 0.5 | 3 | 0.9 | −15 | 80 |
methadone | 0.03 | 0.05 | 0.9899 | 2 | 2 | 1 | 41 | 12 | 0.2 | −35 | 55 |
methamphetamine | 0.01 | 0.05 | 0.9990 | 2 | 5 | 8 | 10 | 5 | 1 | 14 | 80 |
midazolam 2 | 0.1 | 0.5 | 0.9940 | 2 | 4 | 1 | 3.3 | 5.6 | 0.2 | −25 | 74 |
morphine 2 | 0.1 | 0.5 | 0.9995 | −8 | −3 | −2 | 16 | 2.7 | 10 | 20 | 70 |
norketamine 2 | 0.05 | 0.5 | 0.9998 | 2 | −4 | 1 | 3 | 1 | 0.1 | 11 | 78 |
nordiazepam | 0.01 | 0.05 | 0.9990 | 2 | 1 | 1 | 6 | 4.8 | 2.6 | 3 | 81 |
oxycodone 2 | 0.1 | 0.5 | 0.9899 | 6 | 1 | 5 | 20 | 10 | 17 | 5 | 84 |
oxazepam 2 | 0.1 | 0.5 | 0.9999 | 2 | −1 | 1 | 9 | 2 | 4 | −33 | 66 |
pinazepam | 0.01 | 0.05 | 0.9932 | 2 | 5 | 1 | 5 | 1 | 5 | −10 | 78 |
prazepam 2 | 0.1 | 0.5 | 0.9964 | 2 | 9 | 1 | 9 | 1 | 5 | −20 | 92 |
temazepam | 0.05 | 0.1 | 0.9914 | 2 | 2 | 1 | 17 | 9 | 6.3 | −18 | 79 |
triazolam | 0.01 | 0.05 | 0.9899 | 2 | 2 | 1 | 11.8 | 3 | 0.2 | −32 | 59 |
α-hydroxyalprazolam | 0.05 | 0.5 | 0.9954 | −4 | 7 | 4 | 16.4 | 9 | 15 | −20 | 83 |
α-hydroxyflunitrazepam | 0.1 | 0.5 | 0.9989 | −5 | −3 | 4 | 6.3 | 10 | 5 | −15 | 70 |
α-hydroxymidazolam | 0.02 | 0.05 | 0.9910 | 2 | 5 | 1 | 4.8 | 3 | 0.8 | −20 | 69 |
tramadol | 0.01 | 0.05 | 0.9917 | −13 | 10 | 14 | 4 | 8 | 9 | 7 | 75 |
Substances | Blood (ng/mL) | CSF (ng/mL) |
---|---|---|
Methadone | 280 | 460 |
Cocaine | 69 | 125 |
Benzoylecgonine | 3160 | 4640 |
Lorazepam | 25 | 19 |
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Dimitrova, A.; Croce, E.B.; Iaquinta, G.; Ferri, E.; Faccioli, S.; Chiavacci, G.; Gualco, B.; Focardi, M.; Grassi, S.; Vaiano, F. Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS. Forensic Sci. 2024, 4, 546-554. https://doi.org/10.3390/forensicsci4040036
Dimitrova A, Croce EB, Iaquinta G, Ferri E, Faccioli S, Chiavacci G, Gualco B, Focardi M, Grassi S, Vaiano F. Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS. Forensic Sciences. 2024; 4(4):546-554. https://doi.org/10.3390/forensicsci4040036
Chicago/Turabian StyleDimitrova, Alexandra, Emma Beatrice Croce, Gaia Iaquinta, Elisa Ferri, Simone Faccioli, Giulia Chiavacci, Barbara Gualco, Martina Focardi, Simone Grassi, and Fabio Vaiano. 2024. "Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS" Forensic Sciences 4, no. 4: 546-554. https://doi.org/10.3390/forensicsci4040036
APA StyleDimitrova, A., Croce, E. B., Iaquinta, G., Ferri, E., Faccioli, S., Chiavacci, G., Gualco, B., Focardi, M., Grassi, S., & Vaiano, F. (2024). Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS. Forensic Sciences, 4(4), 546-554. https://doi.org/10.3390/forensicsci4040036